Plasmon resonant particles, methods and apparatus

ABSTRACT

A method and apparatus for interrogating a target having a plurality of plasmon resonant particles (PREs) distributed in the target are disclosed. In the method, a field containing the target is illuminated, and one or more spectral emission characteristics of the light-scattering particles in the field are detected. From this data, an image of positions and spectral characteristic values in the field is constructed, allowing PREs with a selected spectral signature to be discriminated from other light-scattering entities, to provide information about the field. Also disclosed are a novel PRE composition for use in practicing the method, and a variety of diagnostic applications of the method.

[0001] This application claims priority under 35 U.S.C. §120 toProvisional Application Serial No. 60/038,677, filed Feb. 20, 1997,entitled “Preparation and Use of Plasmon Resonant Particles”, which ishereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates plasmon resonant entities (PREs),or particles, to methods of interrogating a field containing PREs, andto apparatus for carrying out the method, and to various applications ofPREs.

BACKGROUND OF THE INVENTION

[0003] There are a number of important commercial and scientificapplications of interrogating a target for information about the target.For example, the aim of analyte diagnostic tests and methods is todetect the presence and/or amount of an analyte (the target). The targetanalyte may be detected by reacting the analyte with a detectablereporter that (i) can bind specifically to the analyte and (ii) isdetectable with suitable detecting tools. The reporter may, for example,be a colored or fluorescence molecule, or a colloidal metal, or areporter such as a radiolabel that requires special film orscintillation equipment for its detection.

[0004] In some diagnostic applications, it is desirable to detectproximity relationships in a target analyte, as evidenced by theinteraction between two proximately located probes on the targetanalyte. This forms the basis of so-called homogeneous assays, where thepresence of an analyte is determined by a detectable probe proximityeffect observed when two distinct probes are brought together on closelyspaced sites on the analyte. As an example, two fluorescent molecules,when brought together, may exhibit a detectable fluorescence quenchingor a non-radiative energy transfer effect that acts to shift the Stokesradius between the excitation and emission peaks.

[0005] A chemical, biochemical, or biological target may be interrogatedby a variety of chemical and spectrographic methods to determinechemical structure, the presence of certain chemical groups, or theenvironment of the chemical groups. Notable among these methods aremagnetic resonance methods for determining chemical structure andchemical group environment, spectroscopic methods, such as UV, IR,Raman, ORD, and CD spectroscopy, for detecting specific chemical groups,and mass spectroscopy for determining structure by fragment molecularweight analysis.

[0006] Surface chemical analysis of a target sample may be carried outby bombarding the surface with high-energy particles, e.g., electrons,and detecting the energy of atoms that are ejected from the surface.Electron Spectroscopy for Chemical Analysis (ESCA) is an example of suchan approach.

[0007] Often it is desirable to establish spatial and/or distancerelationships in a target, generally requiring interrogation bymicroscopy. Light microscopy has the advantage of simplicity, ease ofsample preparation, and the feature that the sample can be examined in a“wet” condition. Its disadvantage is the relatively low resolving power,directly related to the wavelength of the illumination source (in the400-650 nm range) and inversely proportional to the numerical apertureof the lens systems (at best, about 1.4), limiting resolution to severalhundred nm).

[0008] High-energy beam microscopes, such as the transmission electronmicroscope (TEM) and the scanning electron microscope (SEM) can achieveresolution down to the low nm range, but require a high-vacuumenvironment of the target sample, limiting applications with biologicalsamples. Atomic force microscopy (AFM) is useful for interrogatingsurface features of a target sample, also with a resolution in the lownm range. The method is limited to surface effects.

[0009] Radiographic and scintigraphic methods for detecting and/orlocalizing sites of high-energy emission are also widely used. Thesemethods tend to be quite sensitive, being able to detect very lownumbers of high-energy emission events, but suffer from relativelyhigh-cost and poor resolution when target spatial information isdesired.

[0010] Despite the variety of methods currently available, there are anumber of target-interrogation tasks of commercial and scientificinterest that are difficult or impossible with current methods. Amongthese are:

[0011] 1. Detecting single (or only a few) molecular events, such as thepresence of one or a few binding sites, or one or a few enzymic sites ona target. This capability would open up new diagnostic applications,e.g., related to the presence or absence of specific intracellularevents, and reduce the amount of sample material needed for a reliableassay and allow miniaturization of the assay.

[0012] 2. Resolving sub-wavelength distance relationships in abiological target in a natural hydrated state. As noted above,subwavelength resolution by high-energy beam microscopy requires thesample target to be in a desiccated state, precluding the observation ofnatural cellular processes, including subwavelength movement of cellularcomponents, and allows the user to perturb the sample duringobservation.

[0013] 3. Direct spatial mapping of selected target sites on abiological target, such as direct mapping of selected sequences in achromosome for purposes of chromosome mapping. Currently, this type ofinformation is either not practical, or in the case of chromosomemapping, is not possible at high resolution and precise localization ofgene sequences.

[0014] 4. Optical reading of microencoded information. The ability todetect unique patterns of individual reporter groups would haveimportant applications in forensics, information storage, metrology, andsecurity identification microcodes.

[0015] It would therefore be desirable to provide a method and apparatusfor interrogating a field for the type of information outlined abovethat is impractical or impossible to obtain by prior art methods.

[0016] It would also be desirable to apply the method to variousdiagnostics applications, to achieve improved sensitivity, spatial anddistance information, ease of sample preparation, and flexibility in thetype of target sample that can be interrogated.

SUMMARY OF THE INVENTION

[0017] In one aspect, the invention includes a method of interrogating afield having a plurality of PREs distributed therein. The methodincludes the steps of illuminating the field with an optical lightsource, and detecting a spectral emission characteristic for individualPREs and other light scattering entities in the field. From thisinformation is constructed a computer image of the positions and valuesof the emission spectral characteristic of individual PREs and otherlight-scattering entities present in the field, as a basis fordiscriminating PREs with a selected spectral signature from otherlight-scattering entities in the field, to provide information about thefield.

[0018] The illuminating step may be carried out at different frequencybands, where the spectral emission characteristic of individual PREs andother light scattering entities in the field are detected at each suchband.

[0019] Alternatively, the illuminating step may include exposing thefield to a plurality of narrowband pulses of light which vary induration, to detect variations in emitted light intensity produced byvariations in duration.

[0020] In another embodiment, where the field preferably includes atleast some non-spherical PREs, the illuminating step may involveexposing the field to polarized light at different orientations and/ordifferent angles of incidence. The detecting step includes detecting achange in value of a spectral emission characteristic as a function ofincident light polarization orientation or angle of incidence.

[0021] The detecting step may include simultaneously detecting thevalues of a spectral emission characteristic of individual PREs andother light scattering entities in the field at a plurality of definedspectral bands. Alternatively, the spectral emission characteristicvalues of individual PREs and other light scattering entities in thefield may be detected sequentially at a plurality of defined spectralbands.

[0022] The PREs may be formed in or added to the field by metalenhancing nucleation centers in the field, by adding pre-formed PREs toa target in the field, or by making PREs at target sites in the field,e.g., by photolithographic methods.

[0023] The method may be practiced to discriminate PREs with a selectedspectral signature from all other light-scattering entities in thefield. The spectral emission characteristic that is detected, as a basisfor the discrimination, is typically peak position, peak intensity, orpeak width at half intensity of the spectral emission curve, peakhalfwidth in the image plane, and/or polarization or angle of incidenceresponse. Other emission spectral characteristics, such as response topulsed beam illumination, are also contemplated.

[0024] The same spectral characteristics, either alone or incombination, are useful for discriminating (i) PREs from non-PRElight-scattering entities, (ii) one selected type of PRE from another,and (iii) PREs that are interacting through proximity effects fromnon-interacting PREs (typically PRPs).

[0025] In another embodiment, the PREs have a surface localizedfluorescent or Raman-active molecular entities, e.g., Raman-activemolecules, and the detecting includes detecting plasmon-resonanceinduced fluorescence emission or Raman spectroscopy emission from one ormore of said entities.

[0026] The method may be carried out to yield information about (i) thetotal number of PREs of a selected type in a field, (ii) the spatialpattern of PREs having a selected range of values of a selected spectralcharacteristic in the field, (iii) a distance measurement between twoadjacent PREs, particularly PREs separated by a distance less than theRayleigh distance, (iv) a change in the environment of the field, e.g.,dielectric constant, that affects the value of a plasmon resonancecharacteristics, or (v) motion of PREs in the field.

[0027] In another aspect, the invention includes apparatus forinterrogating a field having a plurality of PREs distributed therein,for example, in practicing the above method for interrogating a field.The apparatus includes an optical light source for illuminating thefield, and an optical detector for detecting values of a spectralemission characteristic of individual PREs and other light scatteringentities in the field, when the field is illuminated by the lightsource.

[0028] Also included in the apparatus is an image processor operativelyconnected to the detector for constructing, from signals received fromthe detector, a computer image of the positions and detected values ofthe emission spectral characteristic of individual PREs and such otherlight-scattering entities present in the field, and a discriminator fordiscriminating PREs with a selected spectral signature from otherlight-scattering entities in the computer image, i.e., a selected rangeof values of a selected spectral emission characteristic. The apparatusis constructed to display (or store) information about the field basedon the information about the selected PREs.

[0029] One preferred light source is a bright field/dark field lens fordirecting light onto the field. The illumination source mayalternatively be a bright field lens, a dark field lens, a polarizer forproducing polarized-light illumination source, such as a plane-polarizedlight source, a TIR, a pulsed beam, an epi-illumination system in whichlight is reflected by a half-silvered mirror through a dark field/brightfield lens, and a dark field condenser lens. The light source mayinclude means for separately with field with light having differentexcitation wavelengths.

[0030] The optical detector may include structure for spectrallyseparating light emitted from the PREs. The detector in this embodimentoperates to form a computer image of the positions and emission spectralcharacteristic values of individual PREs and such other light-scatteringentities present in the field at each of a plurality of differentemission wavelengths.

[0031] The optical detector may include a two dimensional array ofoptical fibers, a grating or prism for responding to the output of theoptical fibers when aligned to act as a line source of light from thearray, and a two-dimensional detector array for responding to thespread-out spectral light from each fiber in the line source of light.

[0032] The image processor may operate to construct an image of fieldpositions and associated values of peak position, peak intensity, orpeak width at half intensity of the spectral emission curve, peakhalfwidth in the image plane, and/or polarization or angle of incidenceresponse.

[0033] In other embodiments, where the PREs have surface associatedfluorescent or Raman-active molecular entities, the image processoroperates to construct an image of field positions and fluorescence peakof plasmon-resonance induced fluorescence, or a Raman spectral featurein plasmon-resonance induced Raman spectral emission.

[0034] The discriminator may operate to discriminate a selected type ofPRE from all other light-scattering entities in the field, PREs fromnon-PRE subwavelength light-scattering particles, including: (i) PREsfrom non-PRE light-scattering entities, (ii) one selected type of PREfrom another, and (iii) PREs that are interacting through proximityeffects from non-interacting PREs (typically PRPs).

[0035] The information displayed by the apparatus may be related toinformation about (i) the total number of PREs of a selected type in afield, (ii) the spatial pattern of PREs having a selected spectralcharacteristic in the field, (iii) a distance measurement between twoadjacent PREs, particularly PREs separated by a distance less than theRayleigh distance, (iv) a change in the environment of the field, e.g.,dielectric constant, that affects a plasmon resonance characteristics,or (v) motion of PREs in the field.

[0036] In another aspect, the invention includes a composition ofplasmon resonant particles (PRPs) having one or more populations ofPRPs. The composition is characterized by: (a) the PRPs have a width athalfheight of less than 100 nm; (b) the PRPs in a single population areall within 40 nm of a defined wavelength; (c) at least 80% of the PRPsin the composition satisfying criterion (a) are in one or more of thepopulations and have a spectral emission wavelength in a singlerange >700 nm, 400-700 nm, or <400 nm; and (d) each population has atmost a 30% overlap in number of PRPs with any other population in thecomposition. The composition may be used in practicing the abovetarget-interrogation method, and/or in conjunction with the abovetarget-interrogation apparatus.

[0037] In one embodiment at least 80% of the PRPs in the composition arein one or more of the populations and have a spectral emissionwavelength in the 400-700 nm wavelength range. Also in this embodiment,the particles have a composition formed of a solid silver particle, asilver particle with a gold core, or a particle with a dielectric coreand an outer silver shell of at least about 5 nm.

[0038] In one general embodiment, for use particularly in a variety ofdiagnostic applications, the particles have localized at their surfaces,(i) surface-attached ligands adapted to bind to ligand-binding sites ona target, where the ligand/ligand-binding sites are conjugate bindingpairs, (ii) fluorescent molecules, (iii) Raman-active molecularentities, and (iv) a blocking reagent to prevent non-specific binding,(v) a coating with functional groups for covalent coupling to the PRPs,or (vi) combinations of (i)-(v).

[0039] The localized ligand may be one of a conjugate pair, such asantigen/antibody, hormone/receptor, drug/receptor, effector/receptor,enzyme/substrate, lipid/lipid binding agent and complementary nucleicacids strands.

[0040] The composition may have first and second populations of PRPshaving first and second different surface localized molecules orentities. For use in identifying a target having first and secondligand-binding sites, the first and second surface bound molecules arefirst and second ligands effective to bind to the first and secondligand-binding sites, respectively. As an example, the first and secondsurface-localized molecules are oligonucleotides having sequences thatare complementary to first and second proximate sequence regions of atarget polynucleotide. As another example, the first and secondsurface-localized entities may be Raman-active molecular entities withdifferent Raman spectral characteristics.

[0041] The composition may contain first and second populations of PRPs,each with a different shape, at least one of which is spherical ortetrahedral.

[0042] In still another aspect, the invention includes a diagnosticmethod for use in detecting the presence of, or information about, atarget having a molecular feature of interest. The method includescontacting the target with one or more PREs (preferably PRPs) havingsurface localized molecules, to produce an interaction between themolecular feature and the localized molecules, illuminating the targetwith an optical light source, and determining the presence of orinformation about the target by observing a plasmon resonance spectralemission characteristic of one or more PRPs after such interaction withthe target. The diagnostic methods may be carried out, for example, bythe above target-interrogation method above, using the abovetarget-interrogation apparatus.

[0043] In a general embodiment, the target contains a ligand-bindingsite, and the surface-localized molecule is a ligand capable of forminga ligand/ligand-binding complex with the target. The binding interactionis detected by detecting a plasmon resonance spectral emissioncharacteristic of the complex. The surface localized ligand may be, forexample, a polynucleotide, oligonucleotide, antigen, antibody, receptor,hormone, enzyme, or drug compound.

[0044] In a solid-phase format of the method, the target is washed toremove PRPs not bound to the target through a ligand/ligand-bindinginteraction, before detecting complex.

[0045] In a homogeneous phase of the method, the interaction of thePRE(s) with the target is effective to produce either aplasmon-resonance spectral emission characteristic which isdistinguishable from that of the non-interacting PREs, or separatediffraction centers, where the two PREs have different peak wavelengths.By detecting one of these features, the presence of the diagnosticinteraction can be determined.

[0046] In one homogeneous-phase embodiment, the PRE(s) containsurface-localized fluorescent reporter molecules, and the interaction ofa PRE with the target or with another PRE at the target is effective todetectably alter a plasmon-resonance induced spectral emissioncharacteristic of the fluorescent molecules on the PRE.

[0047] In another embodiment, the PRE(s) contain surface-localizedRaman-active molecular entities, and the interaction of a PRE with thetarget or with another PRE at the target is effective to detectablyalter a plasmon-resonance induced spectral emission characteristic ofthe Raman-active molecular entities on the PRE.

[0048] In still another embodiment, the target has two or moreproximately spaced ligand-binding sites, and the complex that formsincludes at least two proximately spaced PREs that have a spectralemission signature different from that of PREs in the absence of bindingto the target, e.g., a change in the spectral emission curve of thecomplex, where the two PREs have substantially the same peak wavelength.Alternatively, where the two PREs have different peak wavelengths, theindividual PREs may be interrogated at the two different wavelengths,and the distance between PREs determined by the distance between centersof the two diffraction patterns in the image plane. The embodiment maybe practiced, for example, by reacting the target with first and secondpopulations of PREs having surface-localized first and second ligands,respectively, for binding to the first and second ligand binding sites,respectively.

[0049] For use in forming a spatial image of the target, where thetarget has multiple ligand-binding sites, contacting the PREs with thetarget produces binding at multiple sites. The detecting step includesconstructing a spatial image of the sites of PRE attachment to thetarget, which is indicative of the relative spacings of theligand-binding sites in the target.

[0050] One application involves the mapping of closely spaced regions ina polynucleotide, where the detecting includes observing the spacingbetween centers of the diffraction patterns of the PREs in the imageplane of the PREs.

[0051] Another application involves gene mapping, e.g., by binding PREswith different complementary surface-localized oligonucleotides to atarget polynucleotide, with such in an extended condition.

[0052] In another embodiment, for use in detecting target sequencemutations or for sequencing by hybridization, the target is an array ofdifferent-sequence oligo- or polynucleotides. The array is contactedwith one or more PREs having one or more surface-localized testpolynucleotides, under conditions which allow the PRE'ssurface-localized polynucleotides to hybridize with the target arrayoligo- or polynucleotides. After washing the target to remove unboundPREs, a spectral emission characteristic of PREs at each region of thearray is detected, to determine the pattern of polynucleotide binding tothe array.

[0053] In another embodiment, the target is a polynucleotide present asa separated band in an electrophoresis gel, and the contacting iscarried out by exposing the surface of the gel to PREs underhybridization conditions. This method simplifies the Southernhybridization method by eliminating a DNA band transfer step.

[0054] In another general embodiment of the method, the molecularfeature of interest is a molecule which functions catalytically to breaka bond between two atoms in a molecular chain. The PRE reagent in themethod is a pair of PREs linked by said chain, where the linked PREs mayhave a spectral emission spectrum different from that of the individual,i.e., separated, PREs. The contacting is carried out under conditionseffective to cleave the molecular chain. The presence of the cleavingagent is detected by the disappearance of the linked-PRE spectralemission signature, or the appearance of the individual-PRE spectralemission characteristic, or a change in the detected distance betweenthe two PREs.

[0055] In another aspect, the invention includes a composition ofplasmon resonant particles (PRPs) characterized by: (a) the PRPs have awidth at halfheight of less than 100 nm; (b) at least 80% of the PRPs inthe composition satisfying criterion (a) are in one or more of thepopulations and have a spectral emission wavelength in a singlerange >700 nm, 400-700 nm, or <400 nm; and (c) surface localized ligandsadapted to bind to ligand-binding sites on a target, where theligand/ligand-binding sites are conjugate binding pairs, (ii)fluorescent molecules, or (iii) Raman-active molecular entities.

[0056] The invention further includes a variety of PRE compositions andmethods discussed in Section VI of the Detailed Description of theInvention.

[0057] These and other objects and features of the invention will becomemore fully apparent when the following detailed description is read inconjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0058]FIG. 1 is a graph of the relative scattering intensity of twooptically observable plasmon resonant entities with disparate peakscattering wavelengths.

[0059]FIG. 2 is a graph of the relative scattering intensity of fouroptically observable plasmon resonant entities with similar peakscattering wavelengths.

[0060]FIG. 3 is a schematic illustration of one embodiment of adarkfield microscope detection system suitable for the observation ofplasmon resonant entities.

[0061]FIG. 4 is an illustration of a liquid analog to asolid-immersion-lens which may be used to observe plasmon resonantentities.

[0062]FIG. 5 is an illustration of a total internal reflection typesample stage suitable for use in the observation of plasmon resonantentities.

[0063]FIG. 6 illustrates a reflecting brightfield/darkfield lenssuitable for PRE imaging.

[0064]FIG. 7 is a reproduction of a transmission electron microscopeimage of two plasmon resonant particles.

[0065]FIG. 8 is a graph of light intensity as a function of position inthe image plane at two different bandwidths emitted by the plasmonresonant particles shown in FIG. 7.

[0066]FIG. 9 is a graph showing the results of an assay performed withplasmon resonant labels.

[0067]FIG. 10A illustrates a focused light beam having intensity profilecharacteristics measurable with plasmon resonant entities

[0068]FIG. 10B illustrated the placement of a plasmon resonant entitywithin the focused light beam of FIG. 10A.

[0069]FIG. 11 is a Raman signature from a Raman-active PRE.

[0070]FIG. 12 is a chicken skeletal muscle section whose ryanodinereceptors have been labeled with anti-ryanodine PRPs.

[0071]FIG. 13 is a Drosophila polytene chromosomes where a specific genehas been labeled by PRPs.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. Definitions

[0072] The following terms have the definitions given below, unlessindicated otherwise:

[0073] “Plasmon resonant particle” or “PRP” denotes a single piece orfragment of material, e.g., spherical particle, which elicits plasmonresonance when excited with electromagnetic energy. A plasmon resonantparticle can be “optically observable” when it exhibits significantscattering intensity in the optical region, which includes wavelengthsfrom approximately 180 nanometers (nm) to several microns. A plasmonresonant particle can be “visually observable” when it exhibitssignificant scattering intensity in the wavelength band fromapproximately 400 nm to 700 nm which is detectable by the human eye.Plasmon resonance is created via the interaction of incident light withbasically free conduction electrons. The particles or entities havedimensions, e.g., diameters preferably about 25 to 150 nm, morepreferably, about 40 to 100 nm.

[0074] The term “plasmon resonant entity” or “PRE” is used herein torefer to any independent structure exhibiting plasmon resonancecharacteristic of the structure, including (but not limited to) bothplasmon resonant particles (PRPs) and combinations or associations ofplasmon resonant particles as defined and described above. A PRE mayinclude either a single PRP or an aggregate of two or more PRPs whichmanifest a plasmon resonance characteristic when excited withelectromagnetic energy.

[0075] A “field having a plurality of PREs distributed therein” is aone-, two-, or three-dimensional region, for example, a target orportion or region of a target having PREs attached or otherwisedistributed therein, such that the PREs in the field, when illuminatedwith an optical light source, exhibit plasmon resonance.

[0076] A “spectral emission characteristic” refers to a spectralscattering characteristic of a PRE related to the plasmon resonance ofthe PRE, as discussed in Section III. As used herein, “emission”, asapplied to PREs, means scattered light produced or excited by plasmonresonance.

[0077] The “value” of a spectral emission characteristic is thequalitative or quantitative value of the emission feature, e.g., thevalue of the detected peak intensity, peak wavelength, or peak width athalf maximum.

[0078] A “selected spectral signature” refers to a selected range ofvalues of a selected spectral emission characteristic, e.g., a range ofspectral peak intensity values.

[0079] A “computer image of the positions and values of the emissionspectral characteristic” refers to a matrix which associates each regionin a field being interrogated with one or more spectral emissioncharacteristic values or signature measured for a light-scatteringentity in that region. The image may be a matrix of stored values, ormay be an actual image showing the locations of light-scatteringentities in one dimension or plane, e.g., the x-y plane, and theassociated spectral emission value in another dimension, e.g., thez-axis.

[0080] A “ligand” is a chemical species, typically a biochemicalspecies, that is capable of forming a specific, typically high-affinitybond with a “ligand-binding” site or molecule. The ligand/anti-ligandform a conjugate pair that can include, for example, antigen/antibody,hormone/receptor, drug/receptor, effector/receptor, enzyme/substrate,lipid/lipid binding agent and complementary nucleic acids strands.

[0081] A “Raman-active molecular entity” is a molecule, molecularcomplex, or particle, e.g., silicon particle, that displays a Ramanspectroscopic signature, preferably through resonance Raman excitation,when excited by electric fields of a plasmon-resonating particle towhich the molecular entity is attached.

[0082] “Surface-localized” ligands and other species refer to molecularspecies that are attached to a PRE by covalent or other molecularforces, e.g., electrostatic or dispersion forces, or which are embeddedin a shell or other surface coating on a PRE.

II. Plasmon Resonance

[0083] The present invention utilizes one or more of a number ofspectral emission characteristics of conductive plasmon-resonanceparticles (PRPs or PREs) to interrogate a field for a variety of typesof information, including the presence or absence of a target, spatialfeatures of a target, the environment of a target, number and/or spatialdistribution of a selected type of target binding sites, and distancerelationships in the target, as will be detailed in Sections III-VIbelow.

[0084] Plasmon resonant entities (PREs) or plasmon resonant particles(PRPs) scatter incident light, and the resulting scattered light has afrequency spectrum characteristic of the particle. A general theorydescribing the interaction of an incident electromagnetic wave with aspherical particle which successfully predicts this resonant scatteringwas developed early in the 20th century (H. C. Van Ve Hulst, LightScattering by Small Particles, Wyley, N.Y., 1957). In a metallic sphere,the incident electromagnetic field induces oscillations, referred to as“plasmons”, in the nearly free conduction electrons of the metal, andthese plasmons produce an emitted electromagnetic field. For somematerials, and for the optimum choice of particle size, shape, andmorphology, there will be a maximum scattering efficiency at awavelength characteristic of the scattering particle and its surroundingmedium. For some materials, the intensity of the emitted light issufficient for observation under an optical microscope. Silver particlesare the most notable exhibitors of this effect, as the wavelength of theresonantly scattered light can be in the visible region of the spectrum.

[0085] Theoretical calculations correctly predict that the resonantlyscattered wavelength of a spherical particle will increase, or be“red-shifted”, with increasing particle diameter and with increasingdielectric constant of the surrounding material. For sphericalparticles, dipole resonance produces a scattered frequency spectrumhaving a single peak at a wavelength which is dependent on the materialthe particle is made from the size of the particle, the shape of theparticle, the morphology of the particle, and the local environment.Larger particles have a longer dipole scattering peak wavelength, andsmaller particles have a shorter dipole scattering peak wavelength. Thespectrum of scattered light may also contain contributions from aparticle's quadrupole resonance. For a given shape, a resonant particlescatters predominantly in a particular wavelength band depending on thecomposition and size of the particle.

[0086] The conductive portion responsible for the plasmons can take manydifferent forms, including solid geometric shapes such as spheres,triangular parallelpipeds, ellipsoids, tetrahedrons, and the like, ormay comprise spherical, cylindrical, or other shape shells. It is alsotrue that a dielectric sphere of similar dimensions, having silver orgold on its surface will also exhibit plasmon resonances, assuming theshell has a thickness of at least about 3 nm, preferably 5 nm or more.

[0087] It can further be appreciated that contact or near contactbetween two plasmon resonant particles will produce an electromagneticcoupling between the particles, thereby producing an entity withproperties in some ways similar to a single particle having a size equalto the sum of the two particles in contact. Aggregations of many plasmonresonant particles can therefore also exhibit plasmon resonance withcharacteristics dependent on the geometry and nature of theconglomerate.

[0088] Another feature of plasmon creation in a metallic particle is thegeneration of enhanced electric fields in the region near its surface.Interactions between this electric field and nearby materials cansignificantly alter both the scattering characteristics of the resonantparticle and the nearby material. For example, Surface Enhanced RamanSpectroscopy (SERS) exploits the localized plasmon resonance inroughened or particle coated silver films to enhance the Ramanscattering of various materials by as much as six orders of magnitude.In this technique, Raman scattering from the materials of interest isobserved, and the local field generated by the plasmons is used toenhance the intensity of that scattering.

[0089] Referring now to FIG. 1, a graph of the relative scatteringintensity of two PRPs is illustrated, demonstrating that different PREscan have differences in spectral characteristics that are easilydetected. Although the spectra shown in FIG. 1 could be produced byeither individual PRPs or PREs of a more complex structure, it will beassumed that the source of the scattered light spectra illustrated inFIG. 1 is from PRPs for explanatory purposes.

[0090] In FIG. 1, the relative intensity of scattered light in arbitraryunits is plotted against wavelength in nanometers. The individualspectra of two different PRPs are shown—one, spectrum 3, having a peakemission 5 at approximately 460 nm, and a second, spectrum 7, having apeak emission 9 at approximately 560 nm. In this figure, the lightintensity of the light emitted by each of the two PREs were individuallynormalized to 1.0. The shape of each spectrum is approximatelyLorentzian, with a width at half maximum of approximately 30 nm for theparticle with 470 nm peak, and approximately 50 nm for the particle with560 nm peak. As has been mentioned above, the light emitted byindividual PRPs can be visually observed with an appropriate opticalmicroscope. If the two PRPs with emission spectra illustrated in FIG. 1were so observed, the PRP with peak 5 at 470 nm would appear blue, andthe PRP with peak 9 at 560 nm would appear yellow.

[0091]FIG. 2 shows the spectral emission curves for a population of fourdifferent populations of PREs, each having an approximately homogeneousproperties. The spectra 10, 11, 12, 13 of the four PREs shown in thisfigure have peak emission wavelengths which vary from approximately 460nm to 480 nm. Visually, each of the four PREs which produce the spectrashown in FIG. 2 would appear blue in color. The four particles can bedistinguished, however, on the basis of spectral peak intensity, i.e.,peak height, or on the basis of the different spectral emission curves,for example, by comparing the ratios of peak height to peak width athalf peak height. Other spectral emission characteristics are discussedbelow.

III. Method and Apparatus for Interrogating a Field

[0092] In one aspect, the invention is directed to a method andapparatus for interrogating a field having a plurality of PREsdistributed therein. The method has three parts, in essence: (i)generating data about one or more spectral emission characteristic(s) ofPREs in the field, (ii) from this data, constructing a computer image ofthe PRE positions (regions in a field) and values of the emissionspectral characteristic of individual PREs and other light-scatteringentities present in the field, and (iii) by discriminating PREs withselected spectral characteristics in the image from otherlight-scattering particles in the field, providing information about thefield, e.g., a target in the field.

[0093] A. Spectral Emission Characteristics

[0094] The invention contemplates detecting one or more of several typesof spectral emission characteristics, for generating an image oflight-scattering particles in the field. The spectral emissioncharacteristics of interest may be plasmon-resonance spectral featuresof a single PRP, a shift in spectral emission feature due to theinteraction of two or more PRPs in close proximity, or a fluorescent orRaman spectroscopic feature induced by the enhanced local electricfields interacting with fluorescent, luminescent, or Raman moleculeslocalized on PREs. The most important of characteristics, and the typeof information available from each, are the following.

[0095] Peak wavelength is the wavelength of the peak of the spectralemission curve, that is, the wavelength at which maximum intensityoccurs. Peak wavelengths for the two spectral emission curves shown inFIG. 1 are indicated at 5 and 9, corresponding to wavelength values of470 nm and 560 nm, as described above.

[0096] The peak wavelength value can be determined in one a number ofdifferent ways, seven of which are described here. The implementation ofeach of the methods will be understood from the disclosed method, andfor some of the methods, as discussed below in the description of thelight source and detector in the apparatus of the invention. All ofthese methods are applicable to measuring the spectral curves for aplurality simultaneously. It will be appreciated that some of themethods are also applicable to measuring the spectral curve of eachlight-scattering entity in the field individually, for example, byrastering a photodetector element over the plane of the field.

[0097] (i) The field is illuminated over a range of illuminatingwavelengths, for example, at each of a series of narrowband illuminationwindows through the visible light spectrum. Typically, a filter wheelinterposed between a white light source and the field is employed togenerate the narrowband illumination frequencies.

[0098] (ii) Light emitted from the field is directed through adispersive element, such as a prism, for breaking the emitted light intoseveral narrowband frequencies, which are then each directed to aseparate detector array. As an example, a prism is used to break theemitted light into red, green and blue components, each directed onto aseparate CCD array.

[0099] (iii) Take the emitted field image into a dense bundle of opticalfibers, through a lens that, for example, magnifies eachlight-scattering spot corresponding to a PRE, such that its image fitsentirely in the core diameter of an optical fiber. Each fiber is thenbroken up by a dispersion element into spread out spectrum line ofdifferent frequencies, which is then read by a line of detector elementsin a two dimensional array. Thus each line in the field is read by a2-dimensional array, one array dimension corresponding to the spectralintensity at each of a plurality of frequencies, and the otherdimension, to different positions along an axis in the field. Thisapproach allows for simultaneous reading of a plurality of PREs at eachof a plurality of spectral wavelengths.

[0100] (iv) Illuminate with multiple narrow band light sources, e.g., 3or 4 separate laser lines in the red, green, yellow and blue. Each laseris chopped at a different frequency, typically all under 100 Hz. Theemitted light from the field is detected in a CCD that can be read at100 frames/sec. Computer analysis involving standard techniques is thenused to determine the amount of light of each color impinging on eachpixel in the CCD array, thereby allowing the spectral emission curve tobe constructed.

[0101] (v) The same information may be obtained by routing the scatteredlight through an interferometer, as described for example, in U.S. Pat.No. 5,539,517.

[0102] (vi) It is also a property of plasmon resonant particles that thescattered light undergoes a 180 degree phase shift relative to theincident light as the wavelength of incident light is swept through theresonant peak. At the peak wavelength, the phase difference is 90degrees. This phase shift can be detected, and the peak scatteringwavelength can be determined as that incident wavelength when a phaseshift of 90 degrees is observed.

[0103] (vii) The intensity of PRE light emission at a plurality ofdefined bandwidths can also be determined by exposing the PREs to shortpulses of incident light of varying duration. In particular, it iseffective to use pulses approximating a step function increase ordecrease, that is, with fast rise time or decay time of only 1 or 2femtoseconds. The scattering response of a PRE is that of a forced anddamped oscillator, and near the resonant wavelength, the response of aPRE to narrowband excitation increases as the excitation pulse lengthincreases. Away from the resonant wavelength, the response to narrowbandexcitation is small, and relatively independent of the excitation pulselength. Exposing a PRE to pulses of varying duration, but alladvantageously less than about 500 femtoseconds, at a particularwavelength and noting how long it takes for the emitted energy to reacha steady state value provides information about how close thatparticular wavelength is to the PRE resonant wavelength. By exciting thePREs to several series of duration variable pulses, wherein each serieshas a different peak wavelength, a curve of scattering cross sectionversus wavelength can be generated.

[0104] The peak wavelength generally shifts toward the red (longerwavelengths) as the size of the PRE increases for silver and gold PREs.Peak wavelength values can also provided information about PRE shape.Shape changes from spherical to hexagonal or triangular resultpredominantly a shift of peak wavelength toward the red.Dielectric-shell PRPs, i.e., particles composed of an inner dielectriccore encased in a conductive metal also tend to have longer peakwavelengths than solid metal particles of the same size.

[0105] Peak intensity is the intensity of the peak of the spectralemission curve, and may be expressed as an absolute or relativeintensity value, as in FIG. 2, which shows four PREs with differentrelative peak intensities ranging from less than 3 to greater than 10.The peak intensity value is determined, as above, by one of a variety ofmethods for determining the spectral emission curves of the PREs, withintensity being determined at the peak wavelength.

[0106] The peak intensity will vary with material, morphology and shape.For a particular PRE, the intensity will be a maximum in the pane offocus.

[0107] Width at half peak height is the width, in wavelength units, ofthe spectral emission curve at half peak intensity. This value may bemeasured as an independent spectral characteristic, or combined withpeak spectral intensity to characterize the spectral emission curve, forexample, the ratio of peak intensity/peak width.

[0108] The four curves shown in FIG. 2 illustrate two spectra withrelatively narrow peak widths (curves 10 and 11), and two withrelatively broad peak widths (12 and 13).

[0109] Generally peak width increases with increasing size of the PRE,and changes as the shape of the PRE changes from spherical tonon-spherical shapes in a manner which can be simulated.

[0110] Width in the image plane is the halfwidth of the centraldiffraction region in the Airy pattern in the image plane. All PRPs aresub-wavelength sources of light, and so their spatial image will be anapproximate point spread function with characteristics defined by theoptical system being used. Assuming that the optical system includes aCCD, with a pixel array of photodetecting elements, the width of thecentral diffraction region, which may cover several pixels, is measuredradially from the peak of the center of the diffraction image to theposition in the center of the image where the intensity has fallen tohalf its peak value (assuming a circular image).

[0111] Since the PRPs are subwavelength scatterers, the halfwidth of theintensity pattern as recorded in the image plane will be proportional tothe wavelength of light being scattered. Therefore, for a reasonablysmooth variation in light intensity from a source (such as a Xenon arc),the light is scattered most strongly is at peak intensity, and one canmake a good estimate of peak wavelength by measuring the width of thehalf intensity of the central diffraction region in the image for eachPRP.

[0112] As will be seen below, this spectral characteristic is useful forprecise determination of the positions of PREs in a field, andparticularly for determining the distance between two PREs of differentpeak wavelengths that are more closely spaced than the Rayleighresolution distance. The intensity of the peak of the diffractionpattern in the image plane can be used for focusing the detector lens onthe field, with the maximal value giving the best focus.

[0113] Polarization measures a spectral characteristic, e.g., peakwavelength, peak height, width at half wavelength, or width at half peakintensity in the image plane, as a function of direction of polarizationof light illuminating a PRE field, or the angle of incidence ofpolarized light. The polarization characteristic depends on PRE shaperather than size, and is due to the fact that a non-spherical PRE mayhave more than one resonance, for example, along the directions of themajor and minor axes in an elliptical PRE. In the latter case,illuminating light directed along the major axis would be shifted towardthe red, while that directed along the minor axis, would be shiftedtoward the blue.

[0114] Pulse or time response provides a measure of the number of lightcycles of the illuminating light that are required to “pump up” thescattering to full intensity. PREs have very fast time response(sub-picosecond), and very large pulses of scattered photons can begenerated, the only limitation being the average input power absorbed.They can accept pulses between 5 to 500 femtosecond for drivingtwo-photon processes or second harmonic generation and other higherorder processes.

[0115] As noted above, pulsed or timed illumination measurements aregenerally made by exposing PREs in the field to short pulses of incidentlight of varying duration, to detect peak wavelength. The time to fullresonance, as measured by intensity versus pulse time, also provides ameasure of the quality of the material as a plasmon resonator. Higherquality material is characterized by a narrower width of the resonancesignature, a higher peak intensity, and a longer time to reach themaximum intensity of scattering when illuminated by pulses of light atthe peak wavelength.

[0116] Phase shift is discussed above in the context of determiningspectral peak at 90 degree phase shift. Phase shift can also giveinformation about the response for excitation wavelength away from theresonant peak wavelength.

[0117] Fluorescence emission lifetime can be observed in PRE particleshaving surface-localized fluorescent molecules. The fluorescenceexcitation can be enhanced by the local electric fields generated nearthe surface of the PRE by light within the plasmon resonance peak.Fluorescence emission can also be enhanced if the wavelength of thefluorescence emitted light is within the plasmon resonance peak. Underappropriate conditions, the fluorescence lifetime can be measurablyshortened in this process.

[0118] The method can be used to detect changes in the excitationenvironment of the fluorescent molecules, e.g., proximate interactionswith other molecules or entities.

[0119] Surface enhanced Raman scattering (SERS) relies on the generationof enhanced electric fields in the region near the surface of a PRE.Interactions between this electric field and nearby materials cansignificantly alter both the scattering characteristics of the resonantparticle and the nearby material. Surface Enhanced Raman Spectroscopy(SERS) traditionally exploits the localized plasmon resonance inroughened or particle evaporated silver films to enhance the Ramanscattering of various materials by as much as six orders of magnitude.The SERS performed in accordance with the present invention is confinedsolely to PREs. In this technique, Raman scattering from the materialsof interest is observed, and the local field generated by the plasmonsis used to enhance the intensity of that scattering by many orders ofmagnitude over traditional SERS. When the Raman active molecule has aresonant absorption near peak of the spectral emission curve of the PRE,the additional SERS enhancement is sufficient to make the Raman signalof the PRE-molecule composite detectable, in accordance with the methodof the invention disclosed in Section III. Measuring changes in the PREresonant Raman spectrum can be used to detect alterations, e.g.,binding, in the local environment of the Raman molecule.

[0120] B. Field to be Interrogated

[0121] The field that is to be interrogated, in accordance with themethod and apparatus of the invention, includes a target or targetregion having a plurality, i.e., two or more PREs distributed in thetarget.

[0122] The target may be any target that is suitable for viewing bylight microscopy, including biological cells or tissues; plant or animalparts or cellular aggregates; a solid surface having surface-localizedligand-binding molecules; a fluid sample containing target analytemolecules, particles or cells; biological sample material, such aschromosomal material placed in an extended condition; artificialmonolayer or bilayer membrane substrates; a microfabricated device, suchas an computer microchip; and a microarray, such as a microarray ofoligonucleotide or oligopeptides on a chip.

[0123] Methods for forming PREs and preparing a target having PREsdistributed therein will be discussed in detail below. At this point,three general cases will be briefly considered. First, preformed PREsare added to a target, for attachment at specific target sites. Thetarget may be washed to remove unbound or non-specifically bound PREs.The target may be manipulated before or after PRE binding to achieve adesired configuration, e.g., an elongated chromosome. Second, nucleationsites may be added to the target. After binding to selected locations onthe target, a metal enhancer solution, e.g., silver solution, is addeduntil an appropriately sized PRE is formed. In the third case, PREs areformed by photolithographic methods, e.g., photomasking andphotoetching, on a metal substrate, e.g., silver substrate.

[0124] The types of information which one wishes to determine, byinterrogating the field containing the target and PREs, in accordancewith the invention include: (i) the total number of PREs of a selectedtype in the field, (ii) the spatial pattern of PREs having a selectedspectral characteristic in the field, (iii) a distance measurementbetween two adjacent PREs, particularly PREs separated by a distanceless than the Rayleigh resolution distance, (iv) a change in theenvironment of the field, e.g., dielectric constant, that affects aplasmon resonance characteristics, (v) motion of PREs in the field, (vi)whether two PREs are linked, or (vii) a fluorescence or Raman emissionof molecules or materials attached localized on PREs. Other types ofinformation, are also contemplated, and will be considered in SectionsIV-VI below.

[0125] C. Apparatus of the Invention

[0126]FIG. 3 is a simplified, schematic view of an apparatus 20constructed in accordance with the invention. The target to beinterrogated, here indicated at 22, is supported on a substrate 23 heldon a microscope stage 24 which is selectively movable in the x-y planeunder the control of a stage stepper-motor device, indicated generallyat 26, under the control of a computer 28, which includes othercomputational components of the apparatus as described below.

[0127] The target is illuminated by an optical light source 30 whichdirects illuminating light, typically light in the visible range, and atone or more selected wavelength ranges, onto the target surface. As willbe detailed below, the light source typically includes a means 32 forgenerating light of a given wavelength or spectral frequency, one ormore filters, such as filter 34, for producing a desired frequency bandof illuminating light, and a lens system 36 for focusing the light ontothe target, in a manner to be detailed below.

[0128] Spectral emission light from the target, in this case lightscattered from the target, is directed through lens 56 to an opticaldetector 58. The optical detector functions, in a manner to be detailedbelow, to detect one or more spectral emission characteristics of theindividual PREs in the illuminated portion of the field. The detector istypically a CCD (Charge Coupled Device) array which operates to generateand store an array of optical intensity values corresponding to thearray pixels, as will be detailed below.

[0129] An image processor contained within computer 28 is operativelyconnected to the detector to receive values of light intensity at eachof the detector array positions, under each selected illuminationcondition, e.g., different wavelength or polarization state. The imageprocessor functions to construct a computer image of the positions andvalues of one or more spectral emission characteristics measured by thedetector. Typically, this is done by treating each pixel in the detectorarray as a position point in the illuminated field, and assigning toeach pixel “position” the light intensity value recorded by that pixel.The image generated by the image processor may be a matrix of storednumbers, e.g., position coordinates and associated spectral emissioncharacteristic value(s), or an actual map in which position arerepresented, for example, in an x-y plane, and each measured spectralemission value, represented as a quantity along the z axis, for eachpixel location.

[0130] A discriminator 42 in the apparatus, also forming part ofcomputer 28, functions to discriminate PREs with a selected spectralsignature, i.e., a selected range of values of one or more selectedspectral emission characteristics, from other light-scattering entitiesin the computer image. Examples of the operation of the discriminatorwill be given below.

[0131] C1. Substrate

[0132] As indicated above, the target is supported on a substrate whichis mounted on a microscope stage. Suitable substrates include standardglass slides, cover slips, clear polystyrene, and clear mica asexamples. Other suitable transparent substrates are those associatedwith a TEM grid, including for example, formvar, carbon and siliconnitride. These TEM-associated substrates are all optically transparentat the thicknesses used. Conducting, semiconducting, and reflectingsubstrates are also suitable for PRE applications.

[0133] Another suitable substrate for use in the present invention arethose which may initially appear opaque to the spectral wavelengths ofinterest for PRE observation, but which can be rendered suitable by theapplication of a suitable fluid or vapor. An example is whitenitrocellulose “paper” as used for the transference of biologicalsamples of interest in diagnostic techniques such as “Southerns”,“Northerns”, “Westerns”, and other blotting, spotting, or “dip stick”tests. Once the materials of interest have been transferred and fixed asdesired, the PRE's can be applied as preformed entities, or one canapply PRE nucleation entities and enhance as described below. The whitenitrocellulose at this stage may typically present significantnon-specular light scattering which makes it difficult to visualize thePREs. However, if a suitable treatment which results in a significantreduction of the non-specular scattering is used, for example, allowingacetone vapor to encompass the nitrocellulose substrate, whilemonitoring the PREs, the substrate can become much less opaque, andpermit efficient observation of the PREs.

[0134] Silicon is a preferred substrate for many PRE detectionapplications because it can be made very smooth and free of defects,resulting in very little non-specular scattering under darkfieldillumination. One example of a particularly preferred silicon substrateis the highly polished, etched, and defect free surfaces of siliconwafers commonly used in the manufacture of semiconductors. The nearlycomplete absence of contaminants and surface imperfections of such asubstrate produces excellent contrast of the PRE scattering underdarkfield illumination conditions. However, it should be appreciatedthat such silicon wafers typically have a thin layer of SiO₂ present ontheir surface as a result of the various processing steps. It may bementioned that silicon substrates with approximately 100 nm or more ofSiO₂ on their surface produce some of the most intense, high contrastPRE spectra so far observed from a solid substrate, and it may beadvantageous to intentionally grow a sub-micron layer of SiO₂ on thesilicon wafer surface.

[0135] If the oxide layer is removed from the silicon surface in amanner that prevents rapid re-growth of an oxide layer, for example, byetching in HF acid and passivating the surface with hydrogen, theoptical image of the “point-source” PREs has been observed to betorus-shaped, rather than the usual Airy ring pattern with a brightcentral region. This “doughnut” phenomenon most likely arises as aresult of damping of the transverse driving electric fields (thoseparallel to the silicon surface), leaving only the perpendicular drivingfields which can excite a plasmon mode that radiates well, but not atall directly along the normal. This property of bare silicon substratescan be useful in determining whether a particular PRE is closely boundto the surface of the silicon substrate, or is bound via a tethermolecule or system that has placed it further from the surface, therebychanging the dipole component scattering ratios.

[0136] C2. Light Source and Detector

[0137] With continued reference to FIG. 3, light-generating means 32 inthe light source may suitably be a mercury, xenon, or equivalent arc; ora Quartz-tungsten halogen bulb, of approximately 20 to 250 watts, whichprovides incident light in a frequency band corresponding to wavelengthsfrom approximately 350 nm to 800 nm, for visible light PRE scattering,or a conventional UV source for lower-wavelength PRE scattering.

[0138] Filter 34 typically includes a set of pre-selected narrowbandwidth filters, allowing manual or computer controlled insertion ofthe respective filters. The bandwidth for such filters is typically 5-10nm.

[0139] Other methods of illuminating a target with a series of selectedbandwidths include the use of light sources such as lasers of all typeswhere one may utilize very narrow bandwidths. Multiple frequency sourcesare also contemplated, such as tuned lasers (i.e. Ar-ion) to select anyof the characteristic defined strong “line” sources. Alternatively agrating or prism monochrometer can be used. All the light sources can beeither of continuous or pulsed variety, or a suitable light amplitudemodulation device (not shown) can be inserted in the incident path tovary the intensity level in a prescribed temporal manner. Thepolarization of the light to be incident upon the sample can be variedby the insertion of suitable filters or other devices well known to theart.

[0140] The microscope in FIG. 3 is illustrated to be configured with anepi-illumination system, whereby the collimated light from the sourcefollowing filtering as desired impinges onto a half silvered mirror 38,and is reflected downwards towards the Darkfield/Brightfield (DF/BF)lens 40. In this particular type of DF/BF application, the incidentlight that would have had rays passing through the objective lens isphysically blocked by an opaque circle 42, which is suspended by veryfine webs 44, so as to allow only a concentric band of light to passsuch as bounded radially and illustrated by the rays 46. The unitcomprising mirror 38 and opaque circle 42 may be built into anadjustable block 48 that can be manually (or robotically) moved therebyconverting the microscope from DF/BF to alternate forms of operation.

[0141] Light reflected from the mirror may in turn be refracted orreflected (by a suitable circular lens element 50, fixed to theobjective lens mount into a hollow cone of incident light 52, convergingtoward a focus at the sample plane of the target. As previously noted,the specular reflection of such rays causes them to return along thelines of the incident cone trajectories, where they are ultimatelyabsorbed or otherwise removed from the optical system.

[0142] In this darkfield system illustrated in FIG. 3, the angle betweenthe optic axis and the incident rays illuminating the sample is largerthan the largest angle between the optic axis and the rays scattered bythe PREs which is accepted into the objective lens element 45, which isillustrated to be of the refractive form. Also incorporated in the totaloptical microscope, although not shown, is the ability to divert thelight rays away from detector 38 to other ports whereby the image may beobserved visually through standard binocular eyepieces, or to yetanother port, for example, for photographing the illuminated field.

[0143] It has been found to be suitable to use a Nikon DF/BF lens modelCF Plan BD ELWD with magnification 100X and numerical aperture (N.A.)0.8 as the lens system 54, and also a model CF Plan BD ELWD withmagnification 20X and N.A. 0.4. In that case, the rays entering theobjective element of the lens may be rendered parallel and incident uponthe 50% mirror 38, and into a relay lens 56 (typically magnification of2X or 5X) that focus the rays to an image plane on detector (imagecapture device) 58, where the detection is performed by a suitable CCDcamera system.

[0144] The optical system, including lens 56, is preferably constructedto project the field being viewed into an area corresponding to thearray of the detector, so that each pixel in the array is reading lightfrom a defined region of the field.

[0145] Various image capture devices known in the art may be used,including fiber coupled photodiode arrays, photographic film, etc. Oneexemplary device is a thermoelectrically cooled CCD array camera system,model CH250, manufactured by Photometrics, of Tucson Ariz. This deviceutilizes a CCD chip model KAF1400, having a 1032 by 1037 pixel array.

[0146] It will be appreciated that the detector serves to detect aspectral emission characteristic of individual PREs and otherlight-scattering entities in the field, when the field is illuminated bythe light source, simultaneously at each of the regions in the fieldcorresponding to array pixels.

[0147] C3. Image Processing, Discrimination and Output

[0148] Where the detector is used, for example, to detect spectral peakwavelength, peak intensity, and/or half width of the spectral peak, thedetector measures light intensity at each of a plurality of differentilluminating light frequencies, simultaneously for each of the fieldregions corresponding to a detector array pixel.

[0149] The emission (scattering) values measured at each frequency arestored, allowing spectral emission curves for each region to beconstructed after a full spectrum of illumination. From these curves,peak wavelength, peak intensity, and width at half intensity arecalculated for each region. Similarly, the peak halfwidth in the imageplane can be measured with a CCD array as described above.

[0150] The detector may be supplied with comprehensive software andhardware that allows timed exposures, reading out of the pixels intosuitable files for data storage, statistical analysis, and imageprocessing (as one of the functions of computer 28). This capabilityserves as an image processor for constructing from signals received fromthe detector, first the values of the spectral emissioncharacteristic(s) being determined, and then a computer image of thesevalues and the corresponding associated field positions.

[0151] The image constructed by the image processor may be a matrix ofstored points, e.g., a matrix of associated values of each fieldposition (regions in the field) and values for one or more measuredspectral characteristics, or may be an actual map of field positions,e.g., in the x-y plane, and associated spectral emission values in the zplane.

[0152] The computer in the apparatus also provides discriminator meansfor discriminating PREs with a selected spectral signature from otherlight-scattering entities in the computer image. The basis for thisdiscrimination is noted above in the discussion of various spectralemission characteristics and their correlation with physical propertiesof light-scattering entities.

[0153] Thus, for example, to discriminate PREs with a selected spectralpeak wavelength and peak width at half intensity, the computer imagegenerated could provide a matrix of all field regions and the associatedspectral peak wavelength and width values. The discriminator would thenselected those regions containing PREs whose spectral signature meetscertain ranges of these two spectral emission values. Depending on theparticular values chosen, the discriminator could classifylight-scattering entities in the field in a number of ways, includingdistinguishing:

[0154] 1. PREs with a selected spectral signature from all otherlight-scattering entities in the field;

[0155] 2. PREs from non-PRE light scattering entities in the field;

[0156] 3. For a selected type of PREs, those selected PREs which areinteracting with one another and those which are not; and

[0157] 4. One selected type of PRE from another selected type of PRE inthe field.

[0158] In each case, the basis for the discrimination may be based ondetected values, for each light-scattering entity in the field, of peakposition, peak intensity, or peak width at half intensity of thespectral emission curve, peak halfwidth in the image plane, andpolarization or angle of incidence response. Other spectralcharacteristics mentioned above are also contemplated. In particular,where the PREs have surface-localized fluorescent molecules orRaman-active molecular entities, the detecting may detectingplasmon-resonance induced fluorescent emission or Raman spectroscopyemission from one or more of said molecules or entities, respectively,and these values are used as a basis of discriminating such PREs fromother light-scattering entities. FIG. 1 1 shows a typical Raman spectrumof a Raman-active molecule carried on the surface of a PRE.

[0159] The information obtained from the discriminating step is thenused to provide information about the field. Various types ofinformation available are discussed in Sections IV-VI below. Among theseare:

[0160] 1. The total number of PREs of a selected type in a field. Herethe discriminating step includes counting the number of PREs having aselected range of values of a selected spectral emission characteristicin the constructed computer image;

[0161] 2. Determining a spatial pattern of PREs having a selected rangeof values of a selected spectral characteristic in the field. Here thediscriminating includes constructing an image of the relative locationsof PREs with those spectral-characteristic values;

[0162] 3. The distance between two adjacent PREs, particularly wherethis distance is less than the Rayleigh resolution distance. Here thedetecting includes exposing the field with light of one wavelength, toobtain a diffraction image of PREs in the field, exposing the field withlight of a second wavelength to obtain a second diffraction image ofPREs in the field, and comparing the distance between peaks in the twodiffraction patterns;

[0163] 4. Interrogating a change in the environment of the field. Herethe discriminating includes comparing the values of the detectedspectral characteristic of a PRE in the field before and after thechange, e.g., change in the dielectric of the field;

[0164] 5. Detecting motion of PREs in the field. The detecting hereincludes detecting the centers of the diffraction patterns of the PREsin the image plane, as a function of time.

[0165] C4. Other Embodiments

[0166] Simultaneous imaging of even 100 PRPs or more in the illuminatedfield may be readily and efficiently accomplished, using the apparatusjust described. Alternatively, the apparatus may be designed to “read” aspectral characteristic of each PRE in a field by sequentially scanningeach region in a field with a focused-beam light source, and/orsequentially detecting light scattering from each region in the field,by moving the microscope stage through a small interrogation regiondefined by stationary optics, sequentially interrogating each region todetermine values of a selected spectral characteristics, according toabove-described methods.

[0167] For detecting fluorescent images, the light source is filteredappropriately for the excitation spectrum of the desired fluorophore,and a suitable filter (not shown) is placed in the region between mirror38 and relay lens 56. This filter is chosen to substantially block theexcitation light and permit passage of light in a band matching theemission spectrum of the fluorophore(s) of interest.

[0168] The value of the ability to make comparison of the multipleimages of darkfield PRP treated, brightfield dyed, and/or fluorescentstained samples such as cells and other entities of interest tobiological and medical researchers and clinical applications can bereadily appreciated.

[0169] There are several suitable means for bringing in the incidentlight so as to establish effective darkfield conditions in conjunctionwith suitable means for preferentially and efficiently observing thelight scattered by the PREs. For transparent substrates the incidentlight may be brought in either in transmittance through the substrate,reflectance from the “objective side surface”, or via TIR (totalinternal reflection) at the interface near which the PREs are situated,as shown in FIG. 3 and described in more detail below. In the lattercase the evanescent tail of the TIR light may also be used to excite thePREs, if they are directly outside the reflecting interface, even thoughsuch light field distributions do not radiate directly to the objectivelens.

[0170] For non-transparent substrates, the light must be incident in amanner that results in as near specular reflection as possible, with aminimum of such light reaching the objective. There are several meansfor accomplishing this condition. As one example, incident light may berouted to the field of view through one or more optical fibers. Thefibers may be oriented such that light reflects off the substrate at aglancing angle and does not enter the objective lens of the microscopesystem used to image the PREs. It is also advantageous to usecommercially available DF/BF objective lenses. Examples are those soldby the Nikon Company, Long Island N.Y. An example of the way such aDF/BF lens may be used is illustrated in FIG. 3.

[0171] The objective lens of system in the apparatus can be made aseither a reflecting or refracting lens. For certain PRE applications,especially those requiring the most accurate and rapid focusing of theobjective lens as a function of light wavelength, reflecting lenses maybe preferred because chromatic aberrations can be greatly reduced.Refracting lenses, even those carefully made to compensate for chromaticaberrations when observing light emitted by PREs, can still exhibitsignificant deviation in their frequency dependence of the focal length.The fact that the PREs near the peak of their plasmon resonance can beof such extraordinary brightness for a sub-wavelength sized sourceallows them to be utilized for evaluation of optical components, wherebyone can observe a variety of deviations from the component's ideal“point source” response.

[0172] The use of particular lens types which enhance the numericalaperture of the objective is also contemplated. PREs can be imaged witha standard “solid-immersion” lens having a spherical top and flat lowersurface. Another such contemplated lens is a liquid analog to a solidimmersion lens (SIL) having a fluid between the lower surface of atruncated solid immersion lens and a substrate which has anapproximately equal index of refraction as the lens material.

[0173] Such a lens is illustrated in FIG. 4. The lower flat surface 80of the lens is cut shorter than a standard solid immersion lens. Withthe index matched fluid 82 between the lower surface 80 and a substrate84, the focal plane of the lens is at the usual r/n (where n is theindex of refraction of the lens material) location, which is now insidethe index matched fluid. PRPs in the fluid at this focal plane are thusimaged with this system, allowing focusing on non-substrate bound PREsin solution. Such a lens can readily be added to a commercial DF/BFmicroscope lens by one of ordinary skill in the art, thereby resultingin an improved numerical aperture and increased magnification forviewing samples in a liquid matrix. In particular, this modificationresults in increased brightness and clarity in visualization of thePREs. This configuration allows the user to vary the focal position in aliquid sample while retaining a large numerical aperture and removes theusual requirement of total internal reflection-based illumination as isused for the SIL. This system therefore allows the monitoring ofmovement of a particular PRE in solution.

[0174] In other preferred embodiments, darkfield optics are chosen tooptimize the signal to noise ratio of the PRE signal. This oftenincludes improving the contrast by reducing the background (non-PRE)scattered light to a minimum, or to a minimum relative to the amount ofPRE scattered light observed. For example, using ordinary 1″×3″ glassmicroscope slides, it was determined that the Nikon DF/BF lenses of theextended working distance class had a better contrast for observationusing the 0.8 NA lens compared to the 0.9 NA lens at the samemagnification level (100X), despite the fact that the higher the NA of alens the more scattered light is collected from the PRE. This may beattributed to the change in reflecting properties of the bare glasssubstrate since the angle of incidence of the darkfield illuminationlight is changed when the NA of the objective is changed.

[0175] Polarized incident light can also be used at specific angles(i.e. the Brewster angle) to reduce the amount of reflected light fromthe surface of the substrate. This improves contrast by reducing theamount of non-PRE scattered light which enters the objective lens. Whenimaging non-spherical PREs such as ellipsoidal PREs as described above,the response to plane polarized incident light can also be used todistinguish different PRE populations.

[0176] In another embodiment, a transparent substrate is used which issufficiently thick so as to reduce the amount of light scattered fromits bottom surface that reaches the detector. If particulates arepresent on the bottom surface of a thin glass substrate, some of thescattered incident light will re-enter the detection system and increasebackground. By increasing the thickness of the substrate, one displacesthe region of the spurious scattering further from the optical axis,thereby reducing the amount of non-PRE scattered light that enters thedetector.

[0177] Total internal reflection (TIR) may also be used to illuminatePREs in a transparent substrate from beneath. The evanescent tail ofsuch light can be effectively used to excite the PRE located near thatinterface. There are several methods for exciting PREs with totallyinternally reflected light, such as using an optical fiber whosedimensions and indices of refraction of the inner core and outer layerare chosen so that there is sufficient evanescent field at the fiberoutside surface to excite PREs placed thereon. The light emitted fromthe PRE can be transferred back to the fiber, forming a reflected sourceof light which can be observed by standard methods.

[0178] In another embodiment, illustrated in FIG. 5, a Dove prism 70 isilluminated from the side. A standard glass slide 72 is placed on thetop surface of the Dove prism 70, with a suitable index matching oil 74in between. The incident light 76 is brought in parallel to the majorsurface of the prism 70 and is refracted up toward the slide 72. Theangle of incidence at the slide is selected to be greater than thecritical angle, and results in total internal reflection at the uppersurface of the slide 72. The light then exits the other side of the Doveprism, again parallel to the major face of the prism 70. Evanescentelectromagnetic fields excite PREs bound to the upper surface of theslide, and emit the usual plasmon resonant scattered light into anobjective lens located above the slide.

[0179] This Dove prism geometry is convenient for bringing light fromdiverse sources such as laser, quartz halogen, arc lamp and the like viaan optical fiber or lens to impinge upon the side of the Dove prism, andfilters may be conveniently interposed therebetween to further controlthe nature of the incident light.

[0180] The DARKLIGHT™ light source from Micro-Video of Avon, Mass. canalso be used in a total internal reflection illumination system,although it has been found generally inferior to the Dove prismembodiment described with reference to FIG. 5. This totally internallyreflecting slide illuminator takes light from a halogen source into anoptical fiber, then into the edge of a glass slide. The light undergoestotal internal reflection numerous times while spreading down the slide,exciting PREs with evanescent fields as with the embodiment of FIG. 5.This system is described in detail in U.S. Pat. No. 5,249,077.Additionally, PREs can be observed via the illumination described inU.S. Pat. No. 3,856,398.

[0181] Oil immersion lens systems can also be used in conjunction withTIR illumination. In these systems, though, the index of refraction ofthe medium for internal reflection must be greater than that of the oilbeing used. Accordingly, flintglass, having an index of refraction of1.7, is one preferred prism material.

[0182]FIG. 6 illustrates in cross section a DF/BF objective lens systemcomprising a reflective objective lens 90 combined with a suitableenclosing darkfield illumination ring element 92 as an alternativeillumination scheme. Although the use of such a two component reflectivelens reduces the intensity at the center of the diffraction pattern, thefact that it also substantially reduces the chromatic aberrationcompared to the refractive lenses described in the prior art, makes it apreferred embodiment in conjunction with the means for obtaining thesimultaneous frequency dependent scattering data for a multiplicity ofPREs and for the other non-PRE scattering entities within the field ofobservation that may be imaged and which need to be distinguished andrejected for many applications.

[0183] In some applications, optical microscopy methods must be tailoredto both optically image and analyze the PREs and also to observe thesame PREs and associated sample material with additional instrumentssuch as one or more forms of electron microscopy. In these cases,darkfield optical microscopy must be performed with substrates suitablefor electron microscopy as well. Because the electrons must pass throughthe sample and substrate, the substrate must be very thin, typicallywell under 1 μm. Common substrates include formvar and/or carbondeposited upon a supporting grid. Background scattering is reduced fromthe grid boundaries or “bars” by arranging for the field of applicationof the incident darkfield illumination to be within the spacing of thegrid “bars” and/or to restrict the field of view of the collectingobjective light for the part of the sample under observation. For theBF/DF objective and microscope system, grids with a spacing of up to 400bars per inch and silicon nitride membranes are especially suitable.Alternative forms of darkfield illumination include a separate opticalfiber or lens with darkfield illumination from below the grid substrateand observation of the scattered PRE light from above with a BFobjective lens.

IV. PRP Compositions

[0184] The invention further includes a suspension of plasmon resonantparticles (PRPs) having one or more populations of PRPs. The compositionhas four distinguishing features: (i) the PRPs in each population have aspectral width at halfheight of less than 100 nm; (ii) the PRPs in asingle population are all within 40 nm, preferably 20 nm of a definedspectral emission peak wavelength; (iii) at least 80% of the PRPs in thecomposition are in one or more of the populations and have a spectralemission wavelength in one of the three ranges >700 nm, 400-700 nm; and<400 nm; and (iv) each population has at most a 30% overlap in number ofPRPs with any other population in the composition.

[0185] The first feature addresses the quality of the PRPs, high-qualityemitters being characterized by a relatively narrow frequency range ofscattered light. The second feature provides homogeneity of spectralemission properties for all PRPs within a given population.Specifically, PRPs within a given population all have a peak wavelengthwithin 40 nm of a defined wavelength. The third requirement providesthat a large majority of the PRPs (at least 80% by number) are in one ofthree different wavelength ranges. The fourth requirement defines theuniqueness of the populations, assuming that each population has adistribution of spectral peak wavelengths within 40 nm of a given peakwavelength. The two distribution curves can be no closer than thedistance at which 30 number percent of the particles in one populationfall within the distribution curve defined by an adjacent population.

[0186] Typically, the PRPs in the composition are in one or more of thepopulations, all in the 400-700 nm wavelength range. The PRPs may behomogeneous, e.g., all blue particles, or may be in one of morepopulations, e.g., discrete populations of red, green, and blueparticles, collectively making up 80% of the PRPs in the composition.

[0187] Particles in this spectral range may be formed, as describedbelow, as solid silver particles, silver particle with a gold core, orparticles with a dielectric core and an outer silver shell of at leastabout 5 nm.

[0188] In one general embodiment, particularly for use in a variety ofdiagnostic applications, the particles have localized at their surfaces,(i) surface-attached ligands adapted to bind to ligand-binding sites ona target, (ii) fluorescent molecules, and (iii) Raman-active molecularentities. The ligands are one of the members of a conjugate pair thatcan include antigen/antibody, hormone/receptor, drug/receptor,effector/receptor, enzyme/substrate, lipid/lipid binding agent andcomplementary nucleic acids strands, as examples.

[0189] The PRPs in the composition may have different surface-localizedmolecules on different groups of PRPs. These different groups may bedifferent PRP populations, that is, PRPs with different spectral peakwavelengths, or may be localized on PRPs in a homogeneous PRPpopulation.

[0190] For use in identifying a target having first and secondligand-binding sites, the different surface localized molecules may bedifferent ligands effective to bind to different ligand-binding sites,such as two different-sequence oligonucleotides that bind at differentsequence regions of a common target polynucleotide, or two differentligands that bind to different ligand-binding sites on a macromoleculartarget.

[0191] PRPs may be formed made by a variety of known methods, includingcolloidal chemistry, soluble gel, evaporation/annealing,nucleation/growth via an enhancer, autoradiography, and photoreaction insilver halides and other materials via electromagnetic energy in theform of light, X-rays, or other incident wavelengths. In addition,lithography, electrodeposition, electroplating, and sputtering with ascanning tunneling microscope (STM) tip can be used.

[0192] Where the PRPs have surface localized, e.g., attached ligands,the PRPs are able to bind selectively to a target of interest whichcarries the other half of the ligand/ligand-binding conjugate pair.

[0193] As indicated above, it is also advantageous to produce populationof PRPs having different defined peak wavelength values. When definedpopulations of PRPs are combined with specific binding characteristics,a new class of sub-microscopic probe is created which has significantadvantages over all currently used labeling techniques. The PRPformation techniques described below may be used to create such probes.

[0194] A. Formation of the PRPs By Metal Enhancement

[0195] In this method for forming PRPs, a nucleation center, typically ametal nucleation center in the 1-20 nm size range, is placed at atargeted location, followed by in situ development (enhancing) of thefull conductive body of the PRP. In some applications, a spatiallypre-specified position is designated for the placement of the nucleationcenter. This is in contrast with other applications where nucleationcenters are used to probe a matrix for a particular substance or featurewhose position is not known. Pre-specified placement may be advantageousin metrology and/or instrumentation applications which are furtherdescribed below. For example, it may be desired to place a single PRP onthe end of an optical fiber or a scanning microscope tip. Also, it maybe desired to create a pattern of PRPs in a particular geometricconfiguration.

[0196] Because it is often desirable to create PRPs with controlledspectral characteristics, the enhancing may advantageously be performedwhile being monitored, stopping the process when a PRP having somepre-defined spectral characteristic is formed. This is especiallyconvenient when enhancement chemistry which is not affected by light isused. It is also possible to monitor PR formation by periodicallyterminating the enhancement process, observing the characteristics ofthe entity or entities formed, and re-initiating the enhancement processif one or more spectral characteristics such as color, for example, arenot within a desired range.

[0197] The nucleation center is typically a gold particle 1-10 nm indiameter, and the metal used for enhancing this nucleating site to PRPsize is silver. However, other elements including, for example,platinum, nickel, and palladium, and macromolecules, such aspolynucleotide molecules, are also contemplated as nucleation centersfor the subsequent enhancement process. Non-metallic materials may alsobe used as nucleating centers, such as protein and nucleic acid.

[0198] The configuration of the nucleating center can be controlled soas to produce a PRP having a desired shape or characteristic. Forinstance, triangular or ellipsoidal regions of nucleating material canbe formed. The deposition process may involve many metal depositiontechniques known in the art, such as vapor deposition, sputtering,Ga-focused ion beam (FIB) milling of the thin film prepared of thedesired material, and electroplating into nanopores. Particularly wellcontrolled placement of nucleating material is possible by dischargingnucleating material from the metal tip of a scanning tunnelingmicroscope. Techniques have also been developed whereby individual metalatoms are picked up with the tip of a scanning tunneling microscope,moved, and put down at a desired location. Individual atoms may also be“pushed” to a desired location with a scanning tunneling microscope tip.This technique may be used to place, for example, 10 gold atoms at aspatially pre-specified position for use as a nucleation center.

[0199] In one embodiment, PRPs or PRP nucleating centers are placed at adesired location by introducing the PRP or PRP nucleating center into adrawn micropipette tip, moving the PRP or PRP nucleating center to adesired location and depositing the PRP at this location using standardmicromanipulation techniques. The pipette tips are filled with thedesired material in solutions of varying viscosity (i.e. with gelatin orother matrices), then the tip is manipulated to a selected locationwhile observing under the microscope. By applying pressure to thesolution in the pipette, very small quantities can be deposited onto adesired substrate. Other micromanipulation techniques are also possible.

[0200] Because the PRP material will have a positive dielectric constantat frequencies above the resonant frequency, the metal particles can beoptically trapped in a focused light beam of appropriate wavelengthusing principles similar to currently practiced optical trapping ofplastic particles. The PRP can thus be placed at a spatiallypre-specified position by manipulating the light beam in which it istrapped. Channels of gel may also be created in order to route PRPs tospatially pre-specified positions electrophoretically. If the channel isconfigured to pass proximate to a designated location, a PRP in thechannel will move under the influence of an electric field so as to beguided to the location. Electrostatic bonding techniques can also beused to removably attach PRPs to spatially pre-specified positions.

[0201] In one embodiment, an array of one or more conductive pads ofapproximately 10 to 100 microns in diameter may be created usingstandard integrated circuit lithographic techniques. Each pad mayfurther be selectively connected to a voltage source. As PRPs insolution can be negatively charged, applying a suitable potential to apad will attract a PRP from solution and onto the conductive pad.Removing the voltage and reversing the sign of the applied potential mayfree the PRP if the surface binding forces between the PRP and the padare weak, i.e., approximately one picoNewton.

[0202] Conjugated nucleating centers and/or conjugated PRPs can also beplaced at a spatially pre-specified position by immobilizing the otherhalf of the conjugate pair at the spatially pre-specified position andbinding the conjugate on the nucleating center or PRP to the other halfof the conjugate pair.

[0203] Individual nucleating centers (or fully formed PRPs) may also bedeposited by ejecting droplets of metal particle containing fluid to asubstrate and having the fluid evaporate away by techniques analogous tothose used in ink-jet printing. In this technique, one or more metalparticles can be delivered to a specific location defined by the dropposition. In some embodiments, the concentration of PRPs or nucleatingcenters in the fluid is chosen so that it is statistically likely forone or no PRP or nucleating center to be contained in each drop. Ifnucleation centers are deposited, they may be subsequently silverenhanced to form PRPs. It may further be noted that these techniques canbe used to create a desired geometric pattern of PRPs. When making sucha pattern, drops can be redeposited at those locations where no PRP wasejected during the first pass. Articles with such patterns of PRPs canbe useful in object identification, semiconductor mask registration, andother uses as are described in more detail below.

[0204] As defined herein, the term “in situ” indicates that the PRP isbound to a substrate, immersed in a solution or suspended in a matrix.The definition of “substrate” is discussed below and includes any entitywith which a PRP can associate such as tissue sections, cells, TEM gridsmade from, for example, formvar, silicon (including silicon nitride andsilicon dioxide), mica, polystyrene, and the like. These nucleationcenters are typically colloidal gold preparations, and suitablepopulations of nucleation centers are commercially available either infree form or attached to various biological molecules.

[0205] The silver enhancement of gold nucleating centers to producelarger silver masses which can be visible under an electron and/or alight microscope is currently practiced, and some procedures for imagingbiological systems using silver enhanced gold particles have beenextensively developed. Reagents for performing the enhancement, as wellas the nucleating centers themselves, are accordingly commerciallyavailable. Antibody bound gold nucleation centers are available fromseveral sources, including E-Y Laboratories of San Mateo, Calif., andAmersham of Arlington Heights, Ill. Furthermore, a large body ofliterature describes a variety of suitable methods for performing suchenhancement (see, for example, M. A. Hayat, Ed., Immunogold-SilverStaining—Principles, Methods, and Applications, CRC Press, 1995, thedisclosure of which is hereby incorporated by reference in its entirety,most particularly Chapters 2 and 7, which describe several experimentaltechniques for silver enhancing gold nucleation centers and a discussionof the silver masses formed thereby).

[0206] The enhancement process is preferably performed by mixing goldnucleation centers with a solution of a silver salt such as silveracetate, silver lactate, or silver nitrate, and a reducing agent such ashydroquinone in a citrate buffer at a pH of approximately 3.5 to 3.8. Ithas been suitable to provide a concentration of approximately 6 mM forthe silver salt and approximately 33 mM for the hydroquinone.Commercially available silver enhancement solutions containingappropriate quantities of silver and reducing agents are alsocommercially available from, for example, BBI of the United Kingdom.Those of skill in the art will appreciate that choice of bufferingsystem, silver salt, reducing agent, and other enhancement parametersare preferably optimized for the local environment, target locations,and the like, and that such optimization can be performed without undueexperimentation.

[0207] In accordance with one aspect of the present invention, PRPsuseful in the invention are prepared by silver enhancing nucleatingcenters until the particles possess the properties of plasmon resonantparticles. Most preferably, the silver enhancement parameters arecontrolled such that the PRPs created have pre-defined spectralcharacteristics, such as appearing a particular desired color whenviewed with darkfield microscopy. During enhancement, spectralcharacteristics may be observed for one individual evolving PRP orsimultaneously for a plurality of individual PRPs. Thus, PRPs havingspecific physical properties can be made and placed at a desiredlocation. In addition, PRP nucleating centers can be placed at a desiredlocation, either in situ, in vitro or in vivo, followed by silverenhancement to produce a PRP at the specific location.

[0208] B. Specific Examples of PRP (or PRE) Formation by MetalEnhancement

[0209] PRPs were prepared by silver enhancement of a gold nucleatingcenter as described in the following examples.

EXAMPLE 1 Placement of Gold Colloids on a Substrate

[0210] The Alcian Blue method is one method for attaching gold colloidnucleating centers to a substrate by chemically treating one or morespatially pre-specified positions of the substrate. Alcian blue is apositively charged dye which promotes adhesion of the negatively chargedgold colloids by charge interactions. Portions of the substrate at whichPRPs are undesired may be coated with a blocking agent such as BSA. A100 microliter drop of 100:1 dilution of 5% acetic acid and 2% alcianblue was placed onto a carefully cleaned glass slide for ten minutes.The active site of the substrate was then immersed in doubly distilledwater, rinsed, and dried.

[0211] The gold colloids were diluted to the desired concentration. Thesolution was placed on the alcian blue treated region of the substrateand incubated for 10 minutes. The substrate was then rinsed withdistilled water.

[0212] The gold colloid attached to the substrate was enhanced asdescribed in Example 2.

EXAMPLE 2 Silver Enhancement of Individual Gold Nucleating Centers

[0213] One ml of a 0.1 mg/ml solution of gelatin was mixed with 50 μlinitiator and 50 μl enhancer in an eppendorf tube. The initiator andenhancer were obtained from BBI International (United Kingdom) silverenhancement kit, light microscopy (LM) version, catalog No. SEKL15. Thesubstrate was immediately covered with the enhancer solution and timingwas started. The substrate was then viewed under darkfield illuminationto determine whether PRs were present. The approximate enhancement timefor colloids on glass, silicon or TEM substrates was about one minute,while the approximate enhancement time for conjugate colloids attachedto biological substrates was about seven minutes. These times weredetermined by continuously observing the scattered light from anindividual evolving PRP under darkfield microscopy while the enhancementwas taking place. It can be appreciated that precise enhancementdurations can be utilized to control the scattering response, andtherefore the color, of the PRPs created. Enhancement was stopped byrinsing thoroughly with distilled water.

EXAMPLE 3 Generation of PRPs in Solution

[0214] For uncoated colloids, 100 μl stock gold colloid (BBIInternational, United Kingdom) was added to 20 ml gelatin solution (0.1mg/ml). For protein conjugated colloid, 100 μl stock conjugated goldcolloid was added to 20 ml doubly distilled water with 1%bovine-serum-albumin (BSA) to block the surface. Conjugated colloidsused were bovine serum albumin, goat anti-biotin and rabbit anti-goatIgG.

[0215] Typical gold colloid concentrations are:

[0216] For uncoated colloid:

[0217] 3 nm stock—˜3×10¹³ particles/ml

[0218] 10 nm stock—˜5.7×10¹² particles/ml

[0219] 20 nm stock—˜7×10¹¹ particles/ml

[0220] For protein conjugated colloid:

[0221] 1 nm stock—˜2×10¹⁵ particles/ml

[0222] 5 nm stock—˜1.7×10¹⁴ particles/ml

[0223] 10 nm stock—˜1.7×10¹³ particles/ml

[0224] 20 nm stock—˜2×10¹² particles/ml

[0225] Three drops (150 μl) initiator was then added. Ten μl incrementsof enhancer were then added while stirring, until the amountcorresponding to the desired PRP scattering peak wavelength was added.The PRP resonance was checked by darkfield measurement or by absorptionspectroscopy as described previously.

[0226] C. PRP and PRE Formation With Lithography and Illumination

[0227] Lithographic techniques can be used to specify where a PRP (orPRE) will be formed and to control its shape, shape, morphology andcomposition. Both positive and negative resist methods can be utilizedto lay down either nucleation centers or fully formed PRPs. In thepositive resist method, portions of a layer of resist is removed inorder to form molds into which metal is deposited. After suchdeposition, resist and extra metal is lifted off, leaving a nucleatingcenter or a fully formed PRP behind. In the negative resist method, achosen layer of silver or other suitable metal is covered with a layerof resist. Portions of this resist layer are then polymerized. Whennucleation centers are formed with this technique, the characteristicsize of this polymerized region may advantageously be approximately 5-20nm. For laying down PRPs whose peak wavelengths are in the opticalspectrum, the characteristic sizes of the polymerized regions areadvantageously 40-125 nm. Silver and unpolymerized resist are thenetched away, leaving the metal nucleation centers or fully formed PRPsunder the polymerized portions of the resist. As another alternative,metal forms can be produced which are larger than desired, and materialmay be etched away by ion milling until a PRP of desired characteristicsis formed. All of these techniques are used in the electronics and otherindustries and are well understood by those of skill in the art.

[0228] In addition, metal salts and halides (i.e. in film) can beirradiated to obtain nucleation centers or entire particles. Enhancementcan be performed with the techniques set forth above, or can also beperformed by thermally annealing the metal particles, or may beperformed as is done in photographic development processes, wherein afilm of photochemical metal salts or metal halides is locally irradiatedwith light until PRPs are produced, and the film is then fixed anddeveloped. There are several ways a localized light spot may be producedfor forming nucleating centers or PRPs at desired locations. In oneembodiment, a very localized light spot can be generated using a metalside-coated tapered fiber “near-field” scanning tip to help confine thenucleation to a suitable small size or to grow the silver grainsdirectly. Alternatively, a pre-made special optical fiber tip ofpreferably approximately 150 nm diameter having a PRP on its endconcentrates the light at a desired location to “write” these nucleationcenters onto a photosensitive surface material. In another embodiment, asolid-immersion lens may be utilized to focus the light onto the metalsalts or halides. The solid-immersion lens may include a PRP at itsfocal point in order to further intensify the local radiation of thesubstrate.

[0229] Photochemical silver salts or halides are also sensitive toelectron and ion beam irradiation, as well as irradiation fromradioactive elements. It will be appreciated that these photographicmethods can also be used to produce arrays or patterns of PRPs ofdesired configuration.

[0230] Whether the developing PRPs are in solution, or bound to asubstrate, the enhancing process can be observed in situ with darkfieldmicroscopy and the process stopped once the PRP has reached the desiredsize which corresponds to a particular color. During light sensitiveenhancement procedures, the progress of the enhancing process can beobserved by washing out the enhancer, observing the light scatteringproperties of the particles created, and re-initiating enhancement untilPRPs with desired spectral characteristics are obtained. In analternative embodiment, a relatively light insensitive enhancer can beused and the enhancing process can be observed under continuousdarkfield illumination and scattering data collection. Of course, oncespecific protocols have been developed which indicate enhancer amounts,incubation times, etc., to produce PRPs with given properties,observation of the enhancement process becomes unnecessary.

[0231] D. Formation of a Conjugated PRPs

[0232] As is shown in Example 3 above, it is possible to enhancenucleating centers which are bound to a biological macromolecule such asan antibody. This enhancement of conjugated gold antibodies can besuccessfully performed even when the conjugated gold/antibody is inaqueous solution and has not been previously bound to an antigen in acell or cell organelle. Surprisingly, it has been found that aftersilver enhancement of free conjugated gold nucleating centers to createPRPs, an appreciable fraction of the conjugate molecules originallypresent on the gold colloid are surface bound to the resulting PRPs.Furthermore, the biological molecule can retain biological activityafter the enhancement process.

[0233] In conjunction with this method, the amount of bound conjugate ona given PRP can be controlled by controlling the size of the conjugatebound nucleation center. For example, a commercially available 1 nmdiameter nucleation center may have only one conjugate molecule, and besilver enhanced to form a PRP with that conjugate molecule attached onthe outermost surface thereof. A 20 nm nucleation center will have acorrespondingly larger number of conjugates attached, some of which willend up bound to the surface of the silver enhanced PRP. During silverenhancement, the conjugate bound PRPs can be incubated with a blockingagent such as bovine serum albumin (BSA) to reduce the presence ofnon-specific bonding sites on the surface of the PRP.

[0234] In another embodiment, a conjugate is added to a PRP after thePRP is made. The conjugate associates with the PRP either covalently ornoncovalently. For example, a fully formed PRP is coated with gelatin,agar, polytetrafluoroethylene (Teflon™), PVP, or latex to preventnon-specific charge interactions, followed by covalent attachment of oneor more functional groups thereto by well known methods, thus generatinga PRP attached to a first half of a particular conjugate pair. Thereagents required to couple conjugates to immunogold nucleating centersor formed PRPs are all commercially available.

[0235] This method of forming conjugate bound PRPs also allows controlof the number of conjugate molecules (i.e. “first half” conjugate pairs)bound to the surface of the PRE. In this case, one can incubate barePRPs in a solution of conjugate and a blocking agent such as BSA. Therelative concentrations of conjugate and BSA can be adjusted to produce,on average, the desired amount of conjugate on each PRP. For PRPs ofunusual shape, or comprising concentric shells of different materials,coating with conjugates after formation is typically more convenient.Conjugate bound PRPs which are approximately spherical, however, canconveniently be produced from commercially available conjugate boundgold colloid nucleating centers and silver enhancers as described above.

[0236] Conjugates or other molecules may be bound directly to the metalsurface of the PRP. The surface chemistry involved in such binding iscomplex, but it is currently exploited extensively in many non-PRPimmuno-gold silver staining techniques. Alternatively, the PRPs may becoated with a shell of plastic material such as latex prior to thebinding of additional molecules such as conjugate. Techniques forbinding molecules to latex are also well known. The molecules bound tothe metal directly or plastic shell may be the conjugate itself, or maybe other intermediate reactive groups such as sulfides, amides,phosphates, aldehydes, carboxyl, alcohol, or others to which conjugateor other molecules of interest may be bound. Conjugates or othermolecules of interest may be synthesized onto such a reactive base withknown techniques of combinatorial chemistry.

[0237] E. Formation of PRP Populations with Desired Characteristics

[0238] The differences in emission spectra for the two separate PRPs asshown in FIG. 1 can arise from a number of factors. One significantfactor is size, particles of larger size having resonance peaks atlonger wavelengths, and also having spectral shapes with increasedhalf-maximum widths. Therefore, control of the size of PRPs beingproduced results in control over some spectral characteristics.

[0239] It can thus be appreciated that with the addition of a controlledamount of enhancer, a population of PRPs with a narrow range ofdiameters, and therefore a correspondingly narrow range of resonant peakfrequencies, may be produced, such as is illustrated in FIG. 2 for fourtypes of PRPs. In some advantageous PRP production methods, theparticles can be observed during the enhancement process with a suitablemicroscope. These methods use enhancement chemicals such as aredescribed herein which are relatively unaffected by incident lightneeded to observe development of the PRP during the enhancement process.Thus, PRP development can be observed and halted when it has reached adesired end-point. For those applications in which it may be desirableto use a light sensitive enhancement process, or if development outsidethe microscope is desired, timed sequential enhancement is performed.The samples are rinsed after each application and the status of PRPlight scattering is determined. One can continue with as many sequentialenhancement steps as desired.

[0240] As indicated above, the PRP composition of the invention includesone of more PRP populations having peak wavelengths 40 nm of a definedwavelength. Such homogeneity in PRP population is possible by stepwiseaddition of enhancer coupled with darkfield observation of the PRPcreation as described above. Because the width of a plasmon resonancepeak is typically 20 to 40 nm, it is generally unnecessary to furtherreduce the variance in resonance peaks of the PRP population.

[0241] Populations of PRPs having uniform spectral characteristics canalternatively also be prepared by purifying non-homogeneous populations.PRP conjugates and free PRPs can be separated by conventionalbiochemical methods including column chromatography, centrifugation,electrophoresis and filtration. Because PRPs with surface localizedmolecules or entities can have a significantly different mobility thando free PRPs of the same size, they elute from gel filtration columns ata different rate than do free PRPs. Because PRPs are charged particles,they migrate in an electric field. Thus, PRPs can be manipulated by andeven observed during electrophoresis.

[0242] PRPs having certain desired characteristics can also be separatedbased on their Zeta potentials. Zeta potential separation equipmentsuitable for this use is commercially available (Coulter Corp, Fla.).Radiation pressure may also be used to force PRPs through a matrix atdifferent rates depending on their structural properties. If bound andfree PRPs are subjected to electrophoresis in, for example, an agaroseor acrylamide gel, the free PRPs migrate faster than do the bound PRPs.Likewise, PRP conjugates may be preferentially retained by filters.Purification can alternatively be performed by centrifugation. Thus,with these methods, an original population of PRPs having a wide rangeof spectral characteristics can be separated into subpopulations whichhave a narrower range of spectral characteristics.

[0243] Individual populations of PRPs, may be prepared separately andlater mixed according to a desired combination of PRP property, e.g.,color, in desired amounts, each labeled with the same or differentbiological macromolecules or unlabeled depending on the application. Insuch compositions, it is preferable for the resonance peaks of thedifferent populations of PRPs to be substantially non-overlapping, asdefined above. In some preferred embodiments, the variance in peaklocation of one population of PRPs is controlled to be withinapproximately 20 nm of one defined wavelength, and the variance in peaklocation of another population of PRPs in the mixture is controlled tobe within approximately 20 nm of a second defined wavelength. To avoidsignificant overlap, it is preferable to ensure that the two peakwavelengths are at least 30 to 40 nm apart, and most preferably 50 ormore nanometers apart.

[0244] Applying the above methods, new types of molecular probes can beproduced by binding selected conjugates to selected PRPs. As mentionedabove, a PRP population with a first spectral characteristic can bebound to a first conjugate, and a PRP population with a second spectralcharacteristic can be bound to a second, different conjugate to producetwo differentiable populations of PRPs with different preferentialbinding properties. Such pre-defined mixtures of PRPs are especiallyuseful in improving the accuracy of detection of low abundance moleculesas will be explained further below.

[0245] F. Isolated Non-Spherical and Composite PRPs

[0246] The emission spectra of PRPs is further affected by the detailsof their structure. Ellipsoidal PRPs offer additional parameters foridentification and discrimination. Each ellipsoidal PRP may have two orthree plasmon resonant peaks, depending on whether there is one isotropyaxis or three different principal axis dimensions, respectively.Ellipsoidal PRPs having one isotropy axis show peaks corresponding totwo orthogonally polarized emissions, one associated with plasmonexcitation along the major axis of the ellipse, the other associatedwith plasmon excitation along the minor axis. The distinct plasmonresonance peaks occur at maximal intensity when the polarization ofincident light is along the corresponding principle axis. Thus, theresponse of a fixed ellipsoidal PRP to polarized light may vary with thedirection of incidence.

[0247] A process for making ellipsoidal silver particles consists ofpulling on a glass matrix containing spherical PRPs at a temperaturesuch that the viscosity results in a stretching of the PRPs into prolateellipsoidal particles having a desired aspect ratio. Conditions havealso been described for “pushing” on such particles such that they formoblate ellipsoidal particles. One ellipsoidal PRP containing matrix,Polarcor™ (Corning Company, Corning, N.Y.), consists of alignedellipsoidal PRPs in a glass matrix. This composite material is aneffective polarizer for certain optical frequencies, principally in thered and above. Individual ellipsoidal PRPs contained within such a glassmatrix can be isolated by dissolving the matrix in such a manner so asto not disturb the PRPs. By preparing ellipsoidal gold particles of thecorrect aspect ratio and size in a suitable matrix, then dissolving thematrix in a manner that does not disturb the particles, large quantitiesof ellipsoidal of PRPs having approximately equivalent plasmon resonancescattering properties can be prepared for use in, for example, liquidreagent preparations for biochemical assays as described in detailbelow.

[0248] Other methods can be used to prepare ellipsoidal PRPs, which maybe produced via photoreaction of silver halides or with appropriatelithographic molds. Alternatively, PRPs which are originally formed tobe substantially spherical can be pressed or rolled between two surfacesto flatten them into a desired oblate or prolate ellipsoidalconfiguration.

[0249] Non-spherical and non-ellipsoidal PRPs are observed to havecharacteristic spectral signatures which are different than sphericaland elliptical particles, and which are useful in creating plasmonresonant probes with advantageous size and scattering properties. It hasbeen found that PRPs which scatter strongly in the blue region may beconveniently produced by making spherical silver particles. As discussedabove, increases in particle diameter will red shift the resonant peak.However, for spherical particles, the peak intensity of the scatteredlight begins to drop off as the peak is shifted into the red, andaccordingly strong red scatterers are much more difficult to producewith spherical particles than are strong blue scatterers. Particles ofother geometric shape, however, can produce strong scattering at longerwavelengths. Three such particles are triangular, square, andapproximately hexagonal in cross-section. Triangular, square, andhexagonal silver particles may, for example, be produced viaphotoreaction of silver halides or with appropriate lithographic molds.Isolated hexagonal particles of a similar size as a blue sphericalparticle will typically have a green plasmon resonance peak. Theisolated triangular particles, which may have 50-150 nm characteristicdimension, are of particular interest because they often exhibit aresonance peak in the red part of the visible spectrum. It has beenfound that production of triangular PRPs is one suitable method ofobtaining PRPs which appear red. A specific example of a red triangularparticle and a blue spherical particle are discussed below withreference to FIGS. 7 and 8. It is also possible to bind small sphericalmetal particles into pairs or other conglomerates to form a variety ofplasmon resonant particle shapes.

[0250] In addition, PRPs having concentric shells of dielectric andconductive material can be prepared. In these embodiments of PRPs, thepeak of the plasmon resonance can be tuned to a desired frequency.Specifically, PRPs can be made with the addition of dielectric materialas either the core or external shell tends to red shift the resonantpeak and produces a comparably strong scatterer. For this reason, redPRPs may advantageously be produced with the inclusion of such adielectric shell or core. Particles having a dielectric core and a shellof aluminum have been found to have a plasmon resonance peak in theultra-violet, at approximately 240-280 nm.

[0251] Particles of three layers comprising a dielectric core/metalshell/outside dielectric shell may be useful for further flexibility inchanging the peak response and the scattering strength. In addition, aPRP with multiple concentric conductive shells can be created. Becauseeach shell will have a different diameter, complex scattering spectraoften containing several separate peaks can be produced. These peaks canbe shifted with variations in the dielectric material separating theconductive shells. As will be explained in more detail below, adielectric outer shell, comprising latex, teflon, or other polymercoating, is also useful as a substrate suitable for bindingmacromolecules of interest to the outside of the PRP.

[0252] The production of PRPs having multiple shells of conductivematerial and dielectric can be more complex, but these may bemanufactured with various film deposition techniques including chemicalvapor deposition or sputtering. Other methods for fabricatingmulti-shelled PRP embodiments are described in U.S. Pat. No. 5,023,139to Birnboim et al., mentioned above.

[0253] A PRP having a dielectric core and an outer metal shell can alsobe made with electroless plating techniques. In this process, coreparticles, made, for example, from latex, have their surfaces activatedwith metal atoms which may be platinum atoms. Using enhancementprocedures as described above, these platinum atoms comprise nucleationcenters for silver enhancement and the formation of a shell around thelatex core.

[0254] PRP compositions which may, but do not necessarily include all ofthe limitations (i)-(iv) in the PRP composition just described are alsocontemplated herein, as novel PRP compositions for use in a variety ofapplications discussed herein, including the general method disclosed inSection III.

[0255] Two-ligand composition. The composition contains two populationsof PRPs, each having a different ligand species carried on the PRPsurface. The two ligands are designed to bind to differentligand-binding sites on a target. The two populations PRPs may havedifferent spectral properties.

[0256] Fluorescent-reporter composition. The composition includes PRPshaving surface attached ligands, for binding to the ligand-binding sitesof a target. The composition is a very sensitive, “one-site” reporter,in that fluorescence emission excited by the plasmon resonance spectralemission of the associated particle acts to focus excitation light atthe site of the fluorescent molecules. The composition may also besensitive to the target environment, if such is designed to containfluorescence quenching or fluorescence energy transfer molecules.

[0257] Fluorescent Quenching or Energy Transfer. This compositionincludes two populations of PRPs, each having a surface-attached ligand(which may be the same or different) for binding to two proximate sitesof a target. Each population contains surface-localized florescentmolecules which either produce fluorescence quenching when proximatelydisposed, or which contain donor and acceptor fluorescent molecules fornon-radiative energy transfer when proximately disposed. The compositionis useful, for example, in a homogeneous assay for detecting a targetwith first and second proximate ligand binding sites.

[0258] PRPs with Raman-active entities. The composition includes aplurality of PRP populations, each with a different Raman-active entitylocalized on the PRP surfaces. Each population may contain additionalsurface-localized molecules, e.g., oligonucleotides with different basesequences or combinatorial library molecules, where the identity of eachsurface-localized molecule is associated with a Raman-active entity,e.g., molecule, with a known, unique Raman spectrum signature. Thecomposition is used, for example, to identify combinatorial librarycompounds that are (i) formed on the PRPs according to standardbead-synthesis methods, and (ii) identified as having a desired compoundactivity.

[0259] As another example, the composition is used for chromosomemapping, where the relative spatial positions of known sequence regions,e.g., ESTs or SSTs, are determined by (i) attaching to each PRP with aunique Raman spectral signature, an oligo sequence fragmentcomplementary to one of the chromosome sequences, (ii) hybridizing theprobes with the chromosomal DNA, and (iii) identifying from the uniquespectral signature of each PRP, the relative position of the PRPs boundto the DNA. By placing the DNA in an extended condition, as above, themapping distances separating the sequences can also be determined.

[0260]FIG. 13 shows the binding of an DNA-sequence labeled PRE to aDrosophila polytene chromosomes, illustrating the ability to localizePREs in a chromosome region.

[0261] V. Diagnostic Methods and Compositions

[0262] The diagnostic method of the invention is intended for use indetermining the presence of, or information about, a target having amolecular feature of interest. The method is preferably practiced inaccordance with the method and apparatus described in Section III, andpreferably employing the PRPs described in Section IV, where the PRPshave surface localized ligands.

[0263] In practicing the method, the target is contacted with one ormore PREs having surface localized molecules, to produce an interactionbetween the molecular feature and the localized molecules. Thisinteraction may include (A) binding of a PRE to a target binding site,for example, through a ligand/ligand-binding interaction, to produce aPRE/target complex, (B) binding of two PRPs to closely spaced targetsites, to produce a spectral characteristic evidencing a PRE/PREinteraction, (C) cleavage of a linkage between two PREs, to produceunlinked PREs, (D) binding of a PRE to a target, e.g., through aligand/ligand-binding interaction, to alter the Raman spectrum ofRaman-active molecules on a PRE in a detectable fashion, (E) binding ofa PRE to a target, e.g., through a ligand/ligand-binding interaction, toalter, e.g., quench or enhance the intensity of the fluorescenceemission of fluorescence molecules on a PRE in a detectable fashion, and(F) formation of a linkage between PREs to produce coupled PREs.

[0264] The target is illuminated with an optical light source, in amanner which allows one or more selected plasmon resonance spectralemission characteristics to be determined, as detailed in Section III.The presence of or information about the target by is then determined bydetecting a plasmon resonance spectral emission characteristic of one ormore PREs after such interaction with the target.

[0265] The PREs employed in the method are preferably PRPs constructedas above to contain a surface-localized molecule that is one of aligand/ligand-binding site conjugate pair, such as antigen/antibody,hormone/receptor, drug/receptor, effector/receptor, enzyme/substrate,lipid/lipid binding agent and complementary nucleic acids strands. Wherethe spectral emission characteristic detected is related to a shift orchange in Raman or fluorescence spectral characteristic, the PRPs alsocontain surface-localized fluorescent or Raman-active molecules ofentities, respectively. The PRPs employed may have the quality andhomogeneity attributes of the PRP composition disclosed in Section IV,or may have less stringent uniformity attributes.

[0266] Because PRE probes are extremely sensitive (as noted above, onecan observe and spectrally analyze a single PRE), the method may be madevery sensitive to amount of target analyte (down to one event). Thisallows earlier detection of pathogens in bodily fluids, includingearlier detection of HIV, tumor markers and bacterial pathogens,detection from smaller fluid volumes, and the ability to miniaturizemany existing diagnostic tests.

[0267] A. Binding of a PRE to a Target Binding Site

[0268] In this general embodiment, PREs with surface ligand moleculesare contacted with a target under conditions that lead to PRP-boundligand binding to ligand-binding sites on the target, forming one ormore PRE/target complexes with the target. Typically, the spectralemission characteristic(s) being measured are unchanged by complexformation. That is, neither PRP/PRP proximity spectral emission effectsor changes in spectral emission characteristics caused by PRPinteractions with the target are observed.

[0269] Typically in this embodiment, the target being analyzed isimmobilized or competes for an immobilized binding site. After PRPbinding to the solid phase, immobilized surface, the solid phase iswashed to remove non-bound PRPs before illuminating the target anddetecting a plasmon resonance spectral characteristic of the targetcomplex(es). The PREs contacted with the target may include two or morepopulations, each with different ligands, and preferably each withdifferent spectral signatures associated with different ligands, e.g.,blue particles for one ligand, and red particles for another. As will bedetailed below, this embodiment has applications for:

[0270] (i) detecting the presence of an target analyte, where theanalyte is either immobilized, competes with an immobilized bindingagent, or can be separated from unbound PRPs in the contacting mixture;

[0271] (ii) in situ hybridization of PRP-oligonucleotide conjugates witha DNA target, to isolate PRPs at the site of sequence hybridization;

[0272] (iii) mapping spatial features of the target, for example, thearrangement of a specific binding site on a target cell or tissue, or ina DNA target, for chromosome mapping; and

[0273] (iv) In situ labeling of a target, for example, in a Southernblot, directly binding probe-labeled PRPs to a DNA fractionation gel, toidentify separated DNA bands.

[0274] The following examples illustrate various assays in which PRPswith surface attached ligand molecules are bound to immobilized targettissue, for purposes of detecting spatial features of the binding sites,and/or the density of binding sites.

EXAMPLE 4 Labeling of Ryanodine Receptor in Chicken Muscle with PREs

[0275] Frozen chick intercostal muscle fixed in paraformaldehyde was cutin 2-3 μm sections and transferred to prepared coverslips (Cell Takcoated spots in Pap pen wells). Tissue sections were washed three timesfor 5-10 minutes each time with PBS. Nonspecific binding sites wereblocked by incubation in 3% normal goat serum, 1% gelatin, 0.01% TritonX-100 in PBS for 20 min. Coverslips were washed for 5 min with a 1:3dilution of blocking buffer (working buffer), then incubated in a 1:5dilution of mouse anti-ryanodine monoclonal antibody (34C) in workingbuffer for one hour. Coverslips were then washed 6 times for 3-5 minuteseach time with working buffer, followed by incubation with a 1:40dilution of 5 or 10 nm gold particles conjugated to goat anti-mouse IgG(AuroProbe EM, Amersham) for 30 minutes. Coverslips were washed 3 timesfor 3-5 minutes each with working buffer, then 3 times for 3-5 min withPBS. Samples were washed 3 times for 2 min in doubly distilled water,then silver enhanced for 8 min using 50 μl initiator, 50 μl enhancer(IntenSE M Silver Enhancement kit, Amersham) and 1 ml 0.1 mg/ml gelatin.Samples were washed three times for 3-5 min with doubly distilled water,covered with Gelvatol anti-fade media and visualized under darkfieldmicroscopy. Individual PREs were observed regularly spaced along theZ-lines of the muscle which contain the ryanodine receptors. The resultsare shown in FIG. 12.

EXAMPLE 5 Binding of PREs to DNA on Nitrocellulose

[0276] A 1 cm×3 cm piece of nitrocellulose membrane was cut and the topsurface was marked at one end with a pencil to identify the DNA surface.Biotinylated DNA (100 ng) was pipetted onto one end of the surface ofthe marked nitrocellulose. Non-biotinylated DNA (100 ng) DNA was appliedto the other end as a control. The nitrocellulose was placed in adesiccator to dry the DNA spots, then cross-linked with ultravioletlight. The nitrocellulose, DNA surface up, was placed in a smallparafilm dish and incubated for at least 4 hours in blocking solution(50 mM sodium phosphate, 150 mM NaCl, 2% BSA, 1% Tween-20) at roomtemperature in a 100% humidity chamber to prevent evaporation. A goatanti-biotin immunogold conjugate (Nanoprobes, Inc., Stony Brook, N.Y.;about 1×10⁸ colloids), was added and the incubation continued for 2hours at room temperature. Immunogold conjugates were removed, blockingsolution (500 μl) was added, and the nitrocellulose was washed for onehour at room temperature. Blocking solution was removed and the filterthoroughly rinsed with doubly distilled water. Double distilled water (2ml) was added to the filter which was washed for 30 min. After removalof the water, one ml enhancer solution (1 ml 0.1 mg/ml gelatin, 50 μlinitiator, 50 μl enhancer; enhancer and initiator were from BBIInternational, United Kingdom, Ted Pella Catalog #SEKL15) was placed onthe nitrocellulose for 8 minutes. The enhancer solution was then washedaway thoroughly with distilled water. The nitrocellulose was placed onfilter paper, DNA side up, in a desiccator to dry for one hour, thenplaced on a glass slide, DNA side up, and covered with a glasscoverslip. The slide was placed in an acetone fume chamber for 20minutes or until the nitrocellulose became transparent. PREs were viewedusing a darkfield microscope. A high density of PREs were observed wherebiotinylated DNA was spotted compared to a low density of PREs elsewhereon the nitrocellulose, including where non-biotinylated DNA was spotted.The same experiment also successfully detected biotinylated DNA whenfully formed PREs conjugated to goat anti-biotin were used.

EXAMPLE 6 Binding of PREs to DNA in Polystyrene Cell Culture Dish

[0277] Doubly distilled water (150 μl) and 1 M NaHCO₃ (17 μl) were addedto one row of wells in a 48 well culture dish. Neutravidin (10 μg) wasadded to each dish followed by incubation overnight. All incubationswere performed at 4° C., 100% relative humidity. Neutravidin was removedand the wells were thoroughly rinsed with doubly distilled water. 150 μlof blocking buffer (50 mM sodium phosphate, 150 mM NaCl, 2% BSA, 1%Tween-20) was added to each well followed by a 4 hour incubation.Beginning with the second well, 1 μg biotinylated DNA was added to theblocking buffer. In subsequent wells, 10-fold dilutions of biotinylatedDNA were added (0.1 μg, 0.01 μg, . . . ) and the plate was incubated for4 hours. Biotinylated DNA was removed and the wells were thoroughlyrinsed with doubly distilled water. Blocking buffer (150 μl of thebuffer described above) and approximately 10⁸ goat anti-biotinconjugated immunogold colloids was added to each well and incubated for4 hours. The immunogold colloid was then removed, wells were thoroughlyrinsed with doubly distilled water, and colloids were enhanced byapplying 100 μl enhancer solution to each well for 7 minutes. Enhancersolution was removed, wells were thoroughly rinsed with doubly distilledwater and wells were dried with dust-free compressed air. PREconcentration in each well was determined using darkfield microscopy.The PRE concentration was correlated with the DNA concentration in eachwell. The same experiment also successfully detected biotinylated DNAwhen fully formed PREs conjugated to goat anti-biotin were used.

[0278] Fluorescent in situ hybridization (FISH) may be performed withPREs (PRISH) instead of fluorescent labels. In this method, aPRE-labeled oligonucleotide is incubated with a DNA molecule ofinterest. If complementary sequences exist on the PRE boundoligonucleotides, the bound PREs may be observed. Alternatively, twoPREs, each attached to a different oligonucleotide, are incubated with aDNA molecule of interest. If a genetic deletion associated with aparticular disorder is present and the PREs bind on either side of thedeleted region, they will be much closer together in the deletion versusthe wild type. In this method, bound pairs of PREs may be detected byalterations in scattering parameters, or by observing correlated pairsof PREs of either the same or different spectral characteristics. PRISHallows detection of a smaller defect or selected genomic region due togains in localization. With appropriately conjugated PREs, bound PREsmay be observed at several locations along a strand of nucleic acid,providing information about several sites at one time. Distancemeasurements can also be made, as discussed in detail above. Asillustrated by FIGS. 7 and 8, a 230 nm distance between PREs, whichcorresponds to approximately 640 base pairs, can be easily resolved toan accuracy of only tens of nanometers or less with optical microscopy.In addition, the use of PCR or other enhancement steps is unnecessary incontrast to FISH in which enhancement is usually required, although PCRenhancement can also be used in conjunction with PRE hybridizationtests. Genetic deletions and mutations can also be detected using aligase to join two adjacent strands of PRE coupled nucleic acid. If thePRE coupled strands hybridize in a precisely adjacent manner, denaturingwill result in free strands of nucleic acid coupled to a pair of PREs.These bound pairs may then be observed as described above. If thestrands hybridize at locations which are too close or too far apart, theligase reaction will not occur, and bound pairs will not form. Boundpairs of PREs may also be produced with PCR methods if PREs are coupledto hybridizing strands of nucleic acid, and standard PCR techniques areused to amplify the quantity of target nucleic acid present.

[0279] In all of these procedures, the PREs can be conjugated to theoligonucleotides either before or after the oligonucleotides are boundto the target nucleic acid. Furthermore, such tests can be performed invitro, or in a cell. Selective PRE hybridization is also advantageouslyapplied to screening multiple nucleic acid containing sample wellscomprising a library of different nucleic acid sequences. Such samplewells may be provided on library chip arrays or standard multiwelldishes.

[0280] PREs can also be coated with antibodies for use in assaysanalogous to enzyme-linked immunosorbent assay (ELISA) detection ofvarious macromolecules. In one advantageous embodiment, multi-welldishes (i.e. 96-well microtiter plates) are coated with an antibodyspecific for a molecule of interest. A biological fluid to be tested isthen placed in the wells containing the immobilized antibody. APRE-labeled secondary antibody which binds a different region of themolecule than does the immobilized antibody is added to the wells. Theplate is then read with a plate reader compatible with darkfield opticaldetection. The presence and level of PRE binding indicates the presenceand amount of molecule in the biological fluid.

[0281] In another embodiment, a particular sample can also be visualizedusing multiple populations of PREs, each having a distinct spectralsignature, and conjugated to separate antibodies which recognizedifferent binding sites on a target molecule, or which recognizedifferent target molecules. Alternatively, the PREs are coupled to apolyclonal antibody which recognizes a plurality of epitopes on the sametarget protein. The presence of two spectrally distinct PREs at the samelocation indicates a positive signal, while the separate presence ofeither particle would constitute an incomplete identification and wouldbe rejected. This approach significantly reduces false positive signalsin clinical diagnostic assays.

[0282] Additional advantages of PRE immunoassays include the fact thatthe ability to detect one PRE with a good signal to noise ratio obviatesthe need to amplify the signal by using secondary antibodies or enzymesand their substrates. This further eliminates non-specific background.Moreover, the ability to analyze the sample during processing by opticalmicroscopy allows real time correction of incubation and wash conditionsso as to further optimize signal to noise. PRE assays may beconveniently employed with essentially any target substance and variousbinding partners in direct, sandwich, and other widely used testformats, some of which are described in more detail below. Substancestested for and conjugated to PREs include proteins, nucleic acid,ligands, receptors, antigens, sugars, lectins, enzymes, etc.

EXAMPLE 7 PRP Assay of Goat-Antibiotin

[0283] The wells of a polystyrene multi-well dish were coated withbiotinylated BSA. Regular BSA was added to block any remainingnon-specific binding sites in the wells. Samples of goat-antibiotinantibodies ranging in concentration from 0.06 to 600 picograms (pg) wereadded to individual wells. A control sample having no goat-antibiotinantibodies was also assayed. PRPs bound to rabbit-antigoat antibodieswere then added to each well and incubated. Unbound PRPs were washedfrom the wells, and bound PRPs in each well were observed with adarkfield optical microscope. Light sources in the field of view wereanalyzed according to the discrimination techniques described above, andthe remaining scattering sites were individually counted in each well.The results of this test are shown in FIG. 9. The control sample had onecount remaining after image processing, and is illustrated as the darkbar in FIG. 9. The number of counted PREs over the concentration rangetested varied from 4 at 0.06 pg analyte, to over 1000 at 600 pg analyte.

[0284] Because it is advantageous to perform these assays with one ormore populations of PREs having approximately uniform spectralcharacteristics, it is advantageous to form the PRE labels first underconditions which are conducive to forming such approximately uniformpopulations. As mentioned above, the binding of nucleation centers tobinding sites, followed by metal enhancement and optical observation hasbeen described, but this technique provides very little control over thespectral characteristics of the particles thus created. And even whenthese techniques have been performed, no effort to use the PREscattering characteristics to discriminate background and make a highlysensitive assay has been made or proposed. Accordingly, PRE assaysperformed by first labeling target species with nucleation centers, andthen metal enhancing them to form PREs, (rather than forming the PREsprior to binding) are still improved when light discrimination betweenPRE scatterers and background is performed. Furthermore, assays whichuse pre-formed optically observable sub-wavelength light emitters of anykind have not taken advantage of the technique of individually countingparticles to create a sensitive assay. When spectral and spatialdiscrimination of background is performed, such counting can be usefulfor fluorescent or luminescent bead labels in addition to PRE labels. Aswill be discussed below, fluorescence can be enhanced by local plasmonresonance, and thus PRE enhanced fluorescent beads provide an additionalsub-wavelength light emitting label useful for such assays.

[0285] It can also be appreciated that many variations of these types ofassays may be performed with PRE labels. All of the various types ofimmunosorbent assays which are currently performed using fluorescentmolecule labels may be performed with PREs instead. Sandwich andcompetition assays, for example, may be performed with PREs. In thefirst case, an entity such as an antibody having affinity for a targetsubstance to be detected may be immobilized on the bottom of an assaywell. A test sample including the target substance is added to the well,and the target substance binds to the first entity. A second entity,having affinity for a different portion of the target substance, maythen be added to the well, wherein it binds to the target species.Finally, PREs bound to a third entity having affinity for the secondentity are added to the well, which bind in turn to the second entity.After rinsing, it can be appreciated that PREs will only be bound tosites where the target substance has been previously bound. This test isvery useful when the first and second entities mentioned above areantibodies having affinity to different epitopes on an antigen beingassayed. The third entity, bound to the PREs, may then be ananti-species antibody, rather than being a specific binding partner ofthe target substance.

[0286] In a competition assay, a first entity may be immobilized in anassay well, and both PRE coupled second entities and target substancesare added to the well, wherein the second entity and the targetsubstance compete for binding to the first entity. When unbound PREs areseparated, the number of PREs remaining in the well indicates the extentto which the target substance was able to occupy binding sites. In thistype of assay, the PRE bound second entity may be the same as the targetsubstance, or may be a different substance which also has an affinityfor the first entity.

[0287] Those of skill in the art will recognize that PRE labels may beused to bind to a wide variety of molecular complexes in a wide varietyof ways to produce a sensitive assay. As additional examples, theconjugate on the PRE label may be a specific binding partner of theanalyte being tested for. It may be a specific binding partner of animmobilized analyte/antibody complex. As another alternative, PRE maybind to an immobilized antibody, but only if that immobilized antibodyhas previously bound an analyte molecule. Each of these varioustechniques may be especially suitable in a given assay, depending on thechemical nature of the analyte being tested for.

[0288] Furthermore, it will be appreciated that assays for multipleanalytes can be performed simultaneously using populations of PREshaving different spectral signatures. Populations of PREs differentcolor or different polarization responses can be conjugated so as torecognize different target substances. When introduced into a matrixcontaining unknown concentrations of several different analytes, all ofthe assays set forth herein could be performed on several targetsubstances at once by separately counting the PREs associated with eachdistinctive spectral characteristic.

[0289] PRE probes can also be used to screen in vitro combinatoriallibraries. In some conventional versions of this technique, a drugreceptor is labeled with a fluorophore then mixed with beads, thecollection of which constitutes the combinatorial library, and spreadout on a slide. The presence of a fluorescent bead indicates receptorbinding and the presence of a potential drug bound to the bead. In oneembodiment of the invention, the fluorescent receptor is replaced with aPRE-labeled receptor which increases the sensitivity and photostabilityof the assay, thereby allowing for the possible production of theoriginal combinatorial library on smaller beads and the ability tosynthesize and screen larger chemical libraries.

[0290] The libraries may also be synthesized on microchips, where thepresence of a PRE probe indicates receptor binding. Recent applicationsof combinatorial libraries for improved drug discovery may thus beenhanced by using PRE probes as a method of detection of potentialcandidates. Selectively attached PRE increase the resolution andsensitivity of bio-chip detection schemes.

[0291] In all of these assays, PRE calibration is conveniently performedusing PREs of different spectral characteristics than are used to detectthe target entities. In essence, the assays are calibrated byintroducing a predetermined quantity of PREs having a selected spectralcharacteristic to create a control population of PREs which can bedetected and measured in conjunction with the PREs used for the assayfunction. As one specific sandwich assay calibration example, red PREsmay be conjugated to the target entity being tested for, and a knownamount (but of course much lower than a saturating amount) of this PREconjugated target entity is added to the well along with the sample,either sequentially or simultaneously. After rinsing away unboundconjugated red PREs, antibodies to the target entity are added. Afterrinsing unbound target entity antibodies, blue PREs (for example) whichare conjugated to an anti-species antibody are added which bind to theantibody to the target entity. After rinsing, both red and blue PREs arecounted, and the red PRE count provides a calibration count. In analternative to this sandwich format, a direct binding assay calibrationmay also be utilized, wherein different immobilized antibodies areprovided on the bottom of the sample well, and the calibration PREs areconjugated to a specific binding partner to one of the immobilizedantibodies.

[0292] Assays with PREs can also be performed in cells. Conjugated PREscan be bound to both fixed or free sites in cells and their locationsindividually observed. Well known techniques exist for placement ofparticles into cells, including high pressure bursts which cause theparticles to perforate the cell membrane and electro-perforation inwhich high voltage discharges are used for the acceleration process (thePRE is typically charged prior to the electro-perforation techniques).Apparatus for performing these techniques are available from BioRadLaboratories of Hercules, Calif. PREs and PRE conjugates may also beintroduced into cells by conventional transfection techniques includingelectroporation. PREs can also be placed into cells directly by piercinga cell membrane with a micropipette, and directly injecting one or morePREs into the cell. In a preferred embodiment, the PRE is coated (i.e.latex) by well known methods to protect it from biochemical damage.

[0293] In some advantageous embodiments of PRE assays within livingcells, two populations of differently conjugated PREs are inserted intoone or more cells. The separate conjugates associated with each separatepopulation may be selected to bind to a different epitope on a targetsubstance being manufactured in the cell. After injection into the cell,presence of the target substance will be indicated by PRE pairing, whichis detected using the techniques described above. Depending on thenature of the target substance, it may be desirable to have PREs withsimilar, or disparate spectral characteristics associated with eachconjugate.

[0294] It is advantageous to prepare wells for use with PRE assays whichare suitable for observation with darkfield microscopy. For themulti-well plates to include a substrate suitable for darkfieldmicroscopy, the well bottoms are advantageously manufactured withparticular emphasis on uniformity, smoothness, and cleanliness so as tohinder the formation of light scattering imperfections. Such care iscurrently not taken in the production of standard 96 well dishes. Inaddition, the outside surface under the wells should also be relativelyclean and smooth, as the outside surface also provides a lightscattering surface which can introduce undesired background signals. Insome advantageous embodiments, the surfaces of the wells have less thanapproximately 100, or even less than approximately 10, light scatteringimperfections therein. As an additional method of increasing signal tonoise ratios in these assays, the location of imperfections in a wellcan be documented, and a scattering signal from those locations can beignored when the assay is performed with that particular well.

[0295] Typically, the field of view of the optical microscopes used inthese assays comprises all of or portions of the bottom of the well.Thus, when low levels of analyte are being detected, it can be importantto ensure that a minimum amount of analyte stick to the walls of thewell, rather than to the bottom. It is accordingly advantageous toinclude a blocking agent on the walls of the well during production. Tomake such a well, a dish may be inverted and placed on a solutionincluding a blocking agent such as BSA. If the dish is pushed down intothe solution, or some of the air trapped in the wells is removed bysucking it out with a pipette or capillary, the BSA solution can be madeto contact the walls of the well without touching the bottom of thewell. After this step, the desired antibody or other binding agent isimmobilized on the bottom of the well, and then additional blockingagent may be added to block remaining nonspecific binding sites on thewell bottom.

[0296] Assay methods according to the invention can also be automated,employing, for example, the method and apparatus of the inventiondescribed in Section III. Automated plate readers are currently used forconventional assay techniques, and the principles for a robotic PREplate reader are in some ways similar. As with currently available platereaders, a robotic sample loader may or may not be provided. A roboticPRE assay plate reader would advantageously include sample wells and amicroscope for observing all or portions of the bottom of the wells. Insome embodiments, a very small objective lens, which may beapproximately 2 mm in diameter, is lowered down into the well and closeto the well bottom to obtain a high numerical aperture while imaging aportion of the well bottom. In these embodiments, the PREs may beilluminated from the bottom with total internal reflection off thebottom of a transparent well bottom. As the light is gathered by theobjective, light emitting entities can be detected and discriminatedfrom background using automated image analysis techniques as describedabove. Counting the remaining discriminated particle sources can also beautomatically performed. In some embodiments, the field of view of themicroscope may be a portion of the assay well bottom, and the reader maybe configured to discriminate and count particles in several regions ofthe same assay well until a certain predetermined count is read. Onlyafter this count is reached will the reader move to a different assaywell. This technique will save time by moving quickly from well to wellwhen a large signal is present, and will take the time required toobtain adequate count statistics when low numbers of bound PREs arepresent in the well. The reader may also be configured to performadditional levels of discrimination depending on the count received. Forexample, a first discrimination based on the spatial deviation from theexpected point spread function may be performed for all fields of view,but an additional spectral deviation measurement will be made when a lowcount value is obtained. All of the thresholds for performing variouslevels of discrimination can be preprogrammed into the reader, againinsuring that wells having low PRE counts are analyzed to maximizesignal to noise ratios, while time is saved on wells having a largenumber of counts, where signal levels are already high.

[0297] It will also be appreciated that mercantile kits includingingredients for performing assays described herein may be created havingnovel combinations of ingredients. Advantageously, such kits may includea container of PREs having approximately equal spectral characteristics.The PREs may be conjugated to selected biological molecules such asantibodies or other types of specific binding partners for selectedsubstances. They may be coupled to reactive groups for custom formationof conjugate at a later time. Washing and blocking solutions may also beprovided. A second container of PREs may also be provided forcalibration or multiple assays as described above.

[0298] B. Binding of Two PRPs to Closely Spaced Target Sites

[0299] As discussed above, the spectral characteristics of light emittedby PREs is dependent on their proximity to other PREs. Changes inobserved peaks in emitted frequencies, e.g., peak wavelength, spectralwidth at half intensity, the appearance of more than one peak, andchanges in response to polarized light, etc., can all be observed asPREs approach and move away from one another. These features can even beused to determine the approximate distance between PREs, by measuringthe extent of their interaction.

[0300] Agglutination and aggregation-dependent immunoassays are thusperformed using PRE probes, and have the capability of single moleculedetection. In one embodiment, two antibodies are each attached to a PREprobe having either the same or distinct spectral signatures. Theseantibodies bind to the same biomolecule of interest, but atnon-competitive sites. The distance between the two binding sites willplace the PRE probes in close proximity which are directly detected vianarrow band illumination if the two PRE have separated plasmon resonancefrequencies or if they have the same plasmon resonance frequency, by aunique spectral signature as a result of their interaction. For example,blood serum is added to a tube containing PRE probes which have beencoated with antibodies specific for a particular serum component. Afterincubation, the sample is spread on a glass slide and the frequency ofaggregated (i.e. close proximity) PRE probes is determined. This iscompared to control slides on which the serum would either contain ornot contain the molecule of interest. This technique has application tothe multi-PRE labeling and consequent detection of peptides, nucleicacid oligomers or genes, as well as portions of or whole cells orviruses.

[0301] The measurement of binding constants between two entities iscurrently performed by several procedures. Macroscopic binding can bemeasured directly by, for example, isothermal titration calorimetry.Less direct methods include absorbance, fluorescence or changes incircular dichroism associated with complex formation. One problemassociated with these methods is that a high concentration of materialis required to observe a detectable change in signal, and at these highconcentrations the sample may be essentially 100% complexed, thuspreventing the measurement of a binding constant under these conditions.In a preferred embodiment, the two entities are labeled with PRE probes,equilibrium is reached, and the ratio of free to bound allowscalculation of a binding constant.

[0302] The ability to detect when two PREs are adjacent is alsoimportant for assays of molecular association and dissociation. If twoPREs are associated with suitable conjugate pairs and are mixedtogether, they will bind to form a pair or, if not restricted, highercomplexes. As one example, PREs conjugated to oligonucleotides will formsuch pairs or complexes if the oligonucleotide sequences on differentpopulations of PREs contain complementary sequences, or if the PRE boundoligonucleotide sequences are complementary to separate regions of atarget oligonucleotide also present in the matrix.

[0303] C. Cleavage of a Linkage Between Two PREs

[0304] In this embodiment, a PRE is linked to another PRE thorough acleavable linker, e.g., a peptide, oligonucleotide, oligosaccharide orother chemically or enzymatically cleavable linker. The aim of thelinked composition is to detect single chemical or enzyme cleavageevents, on the basis of an observable spectral change resulting fromlinked PREs becoming individual, unlinked PREs, in accordance with thePart B embodiment.

[0305] More generally, linked pairs of PRPs, are distinguished and, ifthe binding is disrupted, by, for example, enzymatic degradation of apeptide linker between the PREs or denaturation, this is reflected bythe changes in the paired or complex PRE spectra. Operation of an enzymemay be monitored by this technique by observing an increased rate ofcomplex formation or disassociation in the presence of the enzyme. Oneadvantageous application of these methods includes monitoring theoperation of a signal transduction cascade in a cell. Conjugated PREsare selected such that the presence of a product of a signaltransduction cascade either disassociates previously bound PREs or bindsdisassociated PREs. The initiation of the cascade can thus be observedwith optical microscopy in a living cell by observing association ordisassociation of PREs in the cell.

[0306] Each PRE can be coated in such a way to result in a highprobability of bound pairs by coupling with a linker such as a peptideor DNA molecule. As discussed herein, when two PREs with the same PRpeak frequency are very close to each other, frequency shifts,additional resonances and polarization effects occur. If one wishes todetermine whether a specific enzyme is present in solution, a linker isused which is susceptible to degradation by that enzyme. For example,serine proteases can be assayed by using a peptide linker containing aprotease recognition site therein. After proteolysis, the spectra of thebound PREs changes dramatically as the PREs separate. In some cases, thePREs may be spatially separated far enough apart when linked such thatthey do not interact appreciably and retain essentially independentscattering spectra both when linked and unlinked. In this case, pairformation and disassociation can still be observed and measured byevaluating PRE positions with a CCD detector, and observe pairs of PREshaving relative motion which indicates that they are linked.

VI. Additional Compositions and Applications of PREs

[0307] A. Monitoring Local Dielectric Environment

[0308] When a PRE in air is surrounded with a medium having a dielectricconstant different from that of air, scattering parameters may changerelative to the scattering parameters characteristic of the particle inair. This effect is reversible if the dielectric medium is removed. Suchparameters include, for example, a change in intensity or shift inwavelength of the resonant peak, changes in the PREs response topolarized light, and a change in the number of resonant peaks. Shifts inthe resonant peak and intensity are observed following the addition ofliquids of different indices of refraction surrounding the PRE, andafter they are removed by suitable washing steps, the PREs exhibit theirprior characteristics. For many materials which exhibit plasmonresonance, raising the index of refraction of the surrounding mediumwill red shift the resonant peak to a longer wavelength.

[0309] The presence of specific substances of interest or otherperturbations in a sample being tested may therefore be detected bynoting the spectral response of PREs to substances which interact withthe PREs. For example, a suitable sample can be prepared having PREbound to a substrate. Selected molecules may be bound to the PREsurface. The optical scattering parameters (intensity, polarizationdependence, angular dependence, wavelength dependence, etc.) of eachsuch PRE are recorded. The sample is then treated with material whichincludes molecules of interest that selectively bind to the surface ofthe PRE in such a manner that after initial treatment and/or subsequentfurther treatments, the PRE scattering parameters have changed,confirming the local absorption of additional material or desorption ofthe additional or initial material, or other changes in the localdielectric environment. It can be appreciated that the initial PREsample may be prepared as a test “library” or used to screen an“applied” library of proteins, antibodies, etc. These peak

[0310] (D) Shift in Fluorescence Spectrum

[0311] shifts and intensity changes can also be used to detect molecularperturbations such as association and dissociation due to changes in thePRE local dielectric environment.

[0312] Information concerning the properties of a subject matrix canalso be obtained by observing the spectral dependence on the relativepositions of a PRE and a nearby substrate such as a smooth Si surface.For example, having made a record of a PRE location and spectralsignature in a given sample, one could add an enzyme or photolyze abond, resulting in movement of the PRE from the substrate, therebychanging the PRE spatial and/or spectral signature. Indeed, if a pair ofsuch PRE were bound together, and one moved while the other remainedbound to the surface, the resulting spectral signatures would clearlyindicate this event. Coatings on substrates can also be used to providefurther flexibility in creating detection and analysis systems utilizingPREs. For example, a coating can be applied to a substrate which willbind a desired polypeptide or polynucleotide or a blocking coating canbe applied which will block non-specific binding of the PRE conjugates.One suitable coating comprises, for example, one or more layers ofdielectric materials which produce anti-reflection properties. Thecoating may also comprise one or more layers of dielectric materialwhich will produce an enhanced radiation by the PRE of the light thatenters the observing optical system. The coating can also be selected soas to displace the PRE a distance away from the basic substrate surface.A given polarization of the light scattered by the PRE will be inhibitedor enhanced depending on the distance from a reflecting surface. Forexample, if a suitable spacer layer of SiO₂ is placed on the silicon, anice point source image peaked in the center is observed as expected. Ifthe SiO₂ layer is adjusted or another dielectric substance is used,conditions can be found related to the PRE resonant wavelength and thedielectric thickness and material where there is destructive andconstructive interference of the PRE due to superposition of the lightreflected from the substrate (interface) and the top of the dielectriclayer. By using silicon or conducting surfaces, a noticeably differentspectral signature is obtained than if the PRE moves away from contactwith that surface or if a dielectric layer intervenes.

[0313] B. Monitoring Motion

[0314] Three dimensional PRE motion may be directly visualized using twoobservational lenses at right angles to each other, each yielding a twoaxis motion in the plane perpendicular to their respective optical axis.This is particularly suited to motions that are small compared to thedepth of field of the objective lens. If the motion to be observed has acomponent which is large compared to the depth of focus of the objectivelens, only one lens is used for three dimensional motion, whereby the“depth” direction is determined via a feedback signal that keeps the PREintensity in focus on the image plane. The other two dimensions aredetermined in the usual manner. PRE distance from the substrate surfacecan also be monitored in TIR illumination systems by measuring theintensity of the light scattered by the PRE as it changes position. Asthe excitation electric field drops off exponentially with distance fromthe reflecting interface, PRE intensity will decrease as it moves awayfrom the substrate surface.

[0315] Because PRE probes are so bright and so small they can be usedfor real-time determination of velocity and relative motion. Forexample, PREs may be used to monitor dynamic cellular processesincluding motor proteins (i.e. kinesin), cell division, vesicletransport, etc. PRE probes are particularly useful for in vivo temporalexperiments over a broad range of timescales because they do notphotobleach. PREs or precursor gold nucleating centers are attached tolipids which become incorporated into cell membranes. Specific PREconjugates are designed to bind to their pair on cell surface receptorsassociated with the cell membrane. This method allows monitoring of, forexample, ion channel openings. PREs may also be used to monitor movementof actin and myosin within muscle cells. PREs bound to or coated withconjugates can be introduced into cells. The conjugate will then bind toits binding partner within the cytosol, nucleus or on various organellemembranes. Activation of cell receptors, for example, by a particulardrug, can lead to morphological changes in cell structure. PREs withinor on cells can thus be used as an optical assay system for drugdiscovery or receptor activation. Once bound, the PRE can be localizedand its motion observed. PREs may also be used to assay macroscopicmotion. For example, a blood cell may be labeled and observed incirculation. Alternatively, the flow of blood or other liquid may inducea corresponding motion of the PRE. PREs can also be introduced intocells by a Biolistic device (BioRad Inc, Richmond, Calif.) or byelectroporation.

[0316] By labeling any entity of interest with a PRE, the motion of thatentity may be monitored using the detection process described herein byincorporating a suitable real time data acquisition and analysis system.Such a system may determine motion in a three dimensional sense and, ifthe movement is confined to a plane, in a two dimensional sense. Notonly is precise information available about the motion in a specificsystem of interest, but also observable are changes in molecular motionafter drug treatment or other changes in the physical and chemicalenvironment such as alterations in temperature, pH, illumination,electric or magnetic field strength, or a change in concentration of anycompound of interest.

[0317] PREs can also be used to monitor physical motion of moremacroscopic objects. For example, a single PRE placed on an insectfeeler could be used to sense its motion which could be regular or inresponse to an external molecule. This is particularly useful indetecting molecular responses to smell and pheromones. PREs are alsoideal tools for allowing analysis of mechanical motion on a microscopicor sub-microscopic scale. By binding PREs to the components of so called“nanobearings” or other micron sized machine parts, three dimensionalmotion can be visualized and analyzed on nanometer scales. In additionto the added expense of electron microscopes, motion is difficult tocapture via electron microscopy as the electron microscope is a scanningdevice, and the field of view is therefore generated over time withsequential scans, rather than viewed in real time as is possible withoptical microscopy. Furthermore, with electron microscopy, extensivesample preparation is required, in addition to an evacuated measurementarea. These factors severely limit the potential application of electronmicroscopy to real time motion analysis.

[0318] C. Near-Field Effects

[0319] The applications of PREs discussed above have focused on thefar-field observation of light scattered by the PREs. However, becausePREs also generate intense, non-radiative short-range electric fields,they may be used to affect the physical, chemical, and spectroscopicproperties of adjacent molecules in useful ways. The spectroscopictechnique of Surface Enhanced Raman Scattering may be extended toinclude the specific enhancement of only those materials in theimmediate vicinity of the enhancing PRE. For example, PREs may beconjugated to bind to a target having a known Raman signature.Successful binding can be detected by observing the surface enhancedRaman spectra of the target. They can also be useful for locallyenhanced excitation and modified emission of nearby fluorophores.Surprisingly, PREs can produce enhanced emission from even high quantumefficiency fluorophores if the surface of the PRE is placed fromapproximately 1 to 5 nm away from the fluorophore. In contrast, it isgenerally thought that the presence of a metal quenches fluorophoreemission of high quantum efficiency fluorophores. This fact can be usedto create fluorescent labels having a much higher brightness or achanged lifetime, compared to when not so associated. A label whichincludes a plasmon resonant conductive core (such as a silver particleof 40-100 nm diameter) and a non-conductive shell, made, for example,from latex, may be created, wherein the shell has fluorescent orRaman-active molecules embedded on or within it. Preferably, the peak ofthe plasmon resonance has a significant overlap with the efficientexcitation band for the fluorophore or Raman active molecule. When thelabel is illuminated, the plasmon resonance excitation of the core willgreatly enhance the observed fluorescence. In accordance with the abovediscussion, the thickness of the non-conductive shell is preferably lessthan or equal to approximately 5 nm in order to produce fluorescenceenhancement. The plasmon resonant core, selected to resonate at a chosenwavelength, thus dramatically improves label performance over thefluorescent latex particles currently commercially available.

[0320] Ellipsoidal PRE responses can also be advantageously employed inconjunction with fluorescence spectroscopy. Because ellipsoidalparticles simultaneously permit resonance excitation at two or threedistinct frequencies, they are particularly effective for localizedexcitation of a selected fluorophore by one such plasmon resonance, andthen simultaneously effective for effecting the radiation (i.e.emission) of the excited fluorophore at wavelengths corresponding to theother plasmon resonance.

[0321] Thus, targeted PREs can induce very localized spectroscopiceffects, again improving the collection of information aboutsubmicroscopic systems. Similar to the case of fluorescent resonanceenergy transfer (FRET), clustering of PREs gives rise to new opticalproperties including localized and Photonic Band Gap modes, which can beused to advantage in making highly responsive PRE-based detectors ofmolecular binding events.

[0322] D. Metrology and Instrumentation

[0323] Excited PREs can produce localized heating, and an individual PREcan be used to write to a polycarbonate substrate. As individual, highlylocalized light sources, PREs can be useful in precision lithography,photochemistry, or for inducing light activated chemical reactions.

[0324] PREs can also be used as markers in conjunction with all othernon-optical forms of very high resolution microscopy, includingelectron, atomic force, and scanning tunneling microscopy. In theseapplications, a macromolecule of interest, such as a segment of DNA, ismarked with one or more optically observable PREs. Preferably, the highresolution microscope is also equipped with darkfield optical microscopyapparatus for optically observing the portions of the surface to beimaged with the non-optical microscope. The PRE's bound to the moleculecan be optically observed, and the relevant portion of the highresolution microscope, such as the atomic force or scanning tunnelingprobe tip, can be immediately positioned at a location of interest onthe molecule to be observed. This can increase the efficiency of the useof high resolution microscopes, saving excess scanning time normallyused to locate the object to be imaged. Atomic force, scanningtunneling, or any other type of scanning high resolution microscope canadvantageously be constructed to incorporate darkfield microscopysystems in order to utilize this feature of PREs.

[0325] Industrial applications requiring high precision alignment orregistration may also benefit from the use of PREs. One such applicationis the semiconductor manufacturing process, where lithographic masksmust be precisely aligned with the semiconductor wafer being processed.Because PREs can be localized to a precision of a few nanometers or evenless, a comparison of the position of one or more PREs on thesemiconductor wafer with the position of one or more PREs on thelithographic mask can determine the location of the mask relative to thewafer at the nanometer level. As only relative positioning is important,either random or controlled PRE patterns on the mask and the wafer maybe used.

[0326] Another application of the present invention takes advantage ofthe fact that PREs are essentially point sources of optical frequencylight, having a diameter much less than the emission wavelength. Thus,they produce only the point-spread-function pattern characteristic ofthe instrument through which they are viewed, and not an image of theirstructure. This point spread function can be analyzed to detectimperfections in the optical system used to create it. As one example,an angular variation in the intensity of the circular fringes indicatesa lens in the viewing system which has a circumferential asymmetry.Localizing the center of the Airy pattern at two or more emissionwavelengths also evaluates a lens systems for chromatic aberrations.

[0327] The point source nature of PREs can also be used to test anoptical system for its resolution. Using techniques described above forthe placement of individual PREs at specific locations, a calibrationset of PRE pairs can be created with varying distance between the PREs.It can then be determined how close two PREs must be before the centralpeaks of their respective point spread functions overlap to produce asingle non-differentiated peak. To some extent, the same measurement canbe performed by measuring the width of the peak of a single PRE in thefocal plane with the lens system of interest.

[0328] PREs may also be used to profile the intensity distribution offocused light beams, thereby gathering information concerning theproperties of lenses and other optical systems used to produce suchbeams. As illustrated in FIGS. 10A and B, a focusing lens 100 produces alight beam 102 focused to a narrow beam in the lens focal plane. As iswell known in the art, the beam is not focused to a point at the focalplane, but the intensity has an approximately Gaussian intensity as afunction of distance away from the center of the focused beam. Thedetails of the intensity as a function of position in the focal planewill depend on the characteristics of the optical system which producedthe focused beam.

[0329] Referring now to FIG. 10A, a thin transparent plate 104 may beplaced in the beam 102 at the focal plane. The transparent plate 104includes a PRE mounted thereon. Preferably, of course, the peak of theplasmon resonance response of the PRE is selected to approximately matchthe predominant frequency band of the incident light beam 102. It can beappreciated that the intensity of the light scattered by the PRE willdepend on the intensity of the illumination. Accordingly, if the plate104 or beam 102 is moved such that the PRE moves to different locationsin the focal plane, the intensity as a function of position can bedetermined by collecting scattered light with a suitably placedobjective lens of an observing microscope. As with other darkfieldtechniques described above, the objective of the observing microscopemay be placed so that it collects light emitted by the PRE but does notcollect light transmitted through or specularly reflected by the plate104. This system may be used to test the characteristics ofsolid-immersion-lenses, lasers, and other optical systems.

[0330] E. Object Identification

[0331] Still another application of the present invention is thelabeling and identification of paper or plastic items subject to forgerysuch as paper currency or credit cards, or identification badges. Eitherrandom or pre-defined patterns of PREs may be bonded to the surface ofthe item. In advantageous embodiments, the PREs are coated with aprotective layer or film. Later observation of the proper PRE pattern onthe item with microscopy techniques as described above can be used forauthentication purposes. Such authentication can be implemented via apattern recognition system on a computer, allowing for real timeauthentication at point-of-sale terminals, facility entry locations, andthe like. Alternatively, a magnetic strip, bar code, or other datastorage media may be placed on the item (e.g., a credit card) inaddition to the PRE arrangement. A coded version of the PRE array isalso stored in this media, and a match indicates that the item wasvalidly created. Of course, a cryptographic algorithm which produces amatching magnetic code based on the PRE array that cannot feasibly bededuced from the array itself should be used, and such algorithms arewell known in the cryptographic art.

[0332] G. Forensics

[0333] The robustness and easy visibility of PREs also makes them idealfor several forensic applications. Bodily oils, fluids, DNA, etc. whichis present in fingerprints can bind PREs, making the fingerprint easilyvisible under appropriate illumination. Many different goods may also belabeled with PREs to provide traceability. PREs having differentscattering characteristics can be mixed with explosives, food, drugs,poisons or other toxins, etc. The particular PRE could provide sourceidentification. PREs are ideal for this application because of theirresistance to degradation and the ability to detect even singleindividual PREs in a sample.

[0334] H. Identifying Small Molecules in Combinatorial Libraries byRaman Spectrum PREs

[0335] PREs can also be differentiated by the characteristics ofmolecules which are attached to their surface which may be provided inaddition to the one or more conjugate molecules. Surface enhanced Ramanscattering from Raman active molecules adjacent to individual PREs hasbeen reported (Nie and Emory, Science, Volume 275, 1102-1106, 1997). Ifmolecules with different Raman spectra are attached to differentpopulations of PREs, PREs from different populations may be identifiedby their different Raman scattering signatures. Given the wide varietyof Raman molecules available, a large number of differentiable probesare possible which may be particularly useful in conjunction withcombinatorial library techniques. The use of Raman markers may also beused as an alternative way (in addition to four different plasmonresonance wavelengths) to produce four differentiable PRE populations,which would be useful in DNA sequencing techniques which use fourdifferentiable markers, one for each base. Fluorescent molecules mayalso be bound to PREs to provide an additional marker, as canoligonucleotides, which are distinguished by their preferentialhybridization properties rather than spectrally. If desired, PREs havinga conductive resonant core and a non-conductive dielectric shell such aslatex may include embedded fluorescent molecules in the dielectricshell. This label embodiment is discussed in more detail below. It canbe appreciated as well that combinations of different resonantscattering characteristics, different fluorescent markers, and differentRaman markers can be utilized to prepare hundreds or even thousands ofspectrally differentiable probes. For example, a library may includefour different plasmon resonance signatures, four different fluorescentsignatures, and ten different Raman signatures, thereby producing 160different distinguishable probes by different combinations of theavailable spectral signatures. Accordingly, populations of PREs may bedistinguished based on differences in size or shape, or by differencesin material bound thereto.

[0336] Furthermore, known techniques of combinatorial chemistry can beused to simultaneously synthesize a marker molecule and a conjugatemolecule onto PREs in a simultaneous series of molecular assembly steps.In some embodiments, this process would start with a label precursorentity which comprises a PRE having one or more reactive groups bondedto it which may form a base on which combinatorial chemical synthesismay initiate. The reactive groups may include, for example, aphosphates, aldehydes, carboxyls, alcohols, amides, sulfides, aminoacids, or nucleic acid bases. For example, a selected Raman activemolecule could be synthesized simultaneously with an oligonucleotideconjugate. Alternatively, a library of drug candidate compounds may besynthesized simultaneously with identifying oligonucleotide markers.

[0337] I. Cell Sorting

[0338] PRE probes are also suitable for cell sorting, analogous tofluorescent activated cell sorting (FACS). A mixed cell population isanalyzed for one cell type expressing a particular surface antigen usinga particular PRE probe. In addition, several cell types are isolated bysimultaneously using multiple PRE probes because of the number ofuniquely identifiable PRE probes with distinct spectral signatures thatcan be made. It is contemplated that all of the PRE detection andlocalization methods described herein can be fully automated to produce,among other items, cell sorters. With a PRE cell sorter, it isadvantageous to pass the cell population of interest substantially oneat a time into the field of view of a darkfield microscope. Thedetection, discrimination, and analysis techniques described in detailabove can be used in the cell sorting context to identify PRE labelledcells.

[0339] Many different cell routing schemes may be used in such anapparatus. In one advantageous embodiment, the cells are deposited intoa stream of fluid, such as water, which is constrained to move withinthe confines of a surrounding shell of a second fluid, such as an oil,which is substantially immiscible in the first fluid. This forces thecells to remain confined to a small region for darkfield viewing as theypass through the field of view of the microscope. Preferably, theindices of refraction of the two fluids are approximately equal, tominimize reflections of incident light at the interface between them.

[0340] In addition, PRE labeling can be used in addition to, rather thanas a substitute for, fluorescent labeling in a cell sorting technique.In this case, fluorescent labels and PRE labels are made to bind to thesame target cells. The cell sorting may be done based on an observationof the fluorescent marker. If a portion of the sorted cells are saved asan archival record of the result of the sorting process, the PRE can beused to verify successful sorting in the future. This is more effectivethan observing the fluorescence of stored samples, due to the stabilityand non-photobleaching properties of the PRE.

[0341] A further application of the same technology is performed in vivoor ex vivo. In this technique, cells are permeabilized and PRE probe(s)attached to antibodies against a cellular biomolecule of interest areintroduced into the permeabilized cells. The cells are then incubatedwith a combinatorial chemical library. The viable cells are spread outon a slide and the cells are selected which have been “affected” by thechemical library. “Affected” could indicate a change in localization ordistribution of PRE probes due to a change in localization of theattached biomolecule, or it could indicate a clustering of PRE probesleading to a new spectral signature. Because any entity of interest(i.e. cell, DNA, organelle) can be labeled with a PRE, it can then beoptically detected because the collection of PRE can be observed movingas a unit.

[0342] J. Clinical Applications of PREs

[0343] PREs can also be used in a wide variety of clinical applications.One significant area is in the diagnosis of different conditions inanimals, including humans, which can be identified by the selectivebinding of conjugate to specific organs in the animal. In thistechnique, PREs having selected scattering characteristics may beinjected into the bloodstream or ingested by the animal. These PREs mayfurther be bound to an antibody or other conjugate to target or identifythe presence of a particular substance in the animal. Tissue may then beremoved form the animal and tested for the presence of PREs under amicroscope. If desired, control PREs which are not bound to the specificbinding conjugate can also be injected or ingested to determine thenon-specific binding background. These techniques have been developedwith colored latex particles as the probe, and reagents for performingthese tests with the latex particles are commercially available from,for example, Triton Technologies of San Diego, Calif. and MolecularProbes of Eugene, Oreg. The use of PREs, due to their brightness,biocompatibility, and resistance to degradation will improve thesensitivity of such tests.

[0344] Cell modification and therapy techniques such as gene therapy mayalso be enhanced with PREs. In this case, cells having the desiredgenetic characteristics are labeled with PREs and selected with a cellsorter using the techniques set forth above. Selected cells are thenplaced in a patient. If desired, the PRE can be disassociated andremoved prior to placement in the patient.

[0345] Selective heating and drug delivery is also possible with PREs.If PREs are localized in a selected tissue or region of a patient, theycan be illuminated so as to locally heat the tissue or region withoutsignificant affect on neighboring areas of the body. The administrationand activation of light activated drugs is also enhanced with PREs.Light activated drugs can be activated with far less total light energyby being bound to a PRE where the electric field will be enhanced. Theuse of light activated drugs to treat breast cancer has received recentattention, and may be improved by binding the drugs to PREs to enhancetheir activation at locations deeper in the tissue.

[0346] The application of optical PRE detection and analysis tobiochemical systems is considered to provide many advantages overcurrent labeling techniques, and appears to comprise an area where PREanalysis can have a large impact. Other areas, however, may also benefitfrom the PRE detection and spectral analysis of the present invention.

[0347] From the foregoing, it will be appreciated how various objectsand features of the invention have been met. The method and apparatus ofthe invention are ideally suited to a variety of target-interrogationtasks that have been difficult or impossible heretofore, including, asrepresentative examples:

[0348] 1. detecting single molecule events;

[0349] 2. resolving sub-wavelength distance relationships in abiological target in a natural hydrated state;

[0350] 3. direct spatial mapping of selected target sites on abiological target, such as direct mapping of selected sequences in achromosome for purposes of chromosome mapping; and

[0351] 4. optical reading of microencoded information;

[0352] The method and apparatus can further be applied to a wide varietyof diagnostics applications, to achieve improved sensitivity, spatialand distance information, ease of sample preparation, and flexibility inthe type of target sample that can be interrogated.

[0353] Although the present invention has been described with respect toparticular methods, compositions, and devices. It will be appreciatedthat various changes and modifications can b made without departing fromthe invention.

What is claimed is:
 1. A method of interrogating a field having a plurality of PREs distributed therein, comprising illuminating the field with an optical light source, detecting a spectral emission characteristic of individual PREs and other light scattering entities in the field, constructing a computer image of the positions and values of the emission spectral characteristic of individual PREs and other light-scattering entities present in the field, and discriminating PREs with a selected spectral signature from other light-scattering entities based on detected spectral characteristic values unique to the selected-signature PREs, to provide information about the field.
 2. The method of claim 1 , wherein said detecting includes simultaneously detecting the spectral emission characteristic of the light-scattering entities in the field.
 3. The method of claim 2 , wherein said detecting further includes detecting the spectral emission characteristic of the light scattering entities in the field simultaneously at a plurality of defined spectral frequencies.
 4. The method of claim 1 , wherein said illuminating and detecting steps include illuminating said PREs with incident light predominantly in a first frequency band; detecting the spectral emission characteristics of individual PREs and other light scattering entities in the field under illumination at the first frequency band; illuminating said PREs with incident light predominantly in a second frequency band; and detecting the spectral emission characteristics of individual PREs and other light scattering entities in the field under illumination at the second frequency band.
 5. The method of claim 1 , wherein said detecting includes sequentially detecting the spectral emission characteristic of individual PREs and other light scattering entities in the field at a plurality of defined spectral bands.
 6. The method of claim 1 , wherein said illuminating includes exposing the field to a plurality of narrowband pulses of light which vary in duration, and said detecting includes detecting variations in emitted light intensity produced by variations in duration.
 7. The method of claim 1 , wherein at least some of the PREs are non spherical, said illuminating includes exposing the field to polarized light at different orientations and/or different angles of incident, and said discriminating includes detecting changes in a spectral emission characteristic as a function of incident light polarization orientation or angle.
 8. The method of claim 1 , wherein said PREs are formed in the field by a step selected from the group consisting of (i) binding nucleation centers to a field, metal enhancing said nucleation centers, observing enhancement of said nucleation center during said metal enhancing process, and terminating enhancement when a PRE of a desired spectral characteristic has been formed; (ii) adding pre-formed PREs to a target in the field, (iii) making PREs at target sites in the field.
 9. The method of claim 1 , wherein discriminating PREs with a selected spectral signature from other light-scattering entities in the field includes discriminating a selected type of PRE from all other light-scattering entities in the field, based on detected values, for each light-scattering entity in the field, of peak position, peak intensity, or peak width at half intensity of the spectral emission curve, peak halfwidth in the image plane, and polarization or angle of incidence response.
 10. The method of claim 9 , wherein said discriminating is effective to discriminate, for a selected type of PREs, those selected PREs which are interacting with one another and those which are not.
 11. The method of claim 9 , wherein said discriminating is effective to discriminate a selected type of PRE from another selected type of PRE in the field.
 12. The method of claim 1 , wherein the PREs have surface-localized fluorescent molecules or Raman-active molecular entities, and said detecting includes detecting plasmon-resonance induced fluorescent emission or Raman spectroscopy emission from one or more of said molecules or entities, respectively.
 13. The method of claim 1 , for use in determining the total number of PREs of a selected type in a field, wherein said discriminating includes counting the number of PREs having a selected range of values of a selected spectral emission characteristic in the constructed computer image.
 14. The method of claim 1 , for use in determining a spatial pattern of PREs having a selected range of values of a selected spectral characteristic in the field, wherein discriminating includes constructing an image of the relative locations of PREs with those spectral-characteristic values.
 15. The method of claim 14 , wherein the location between two adjacent PREs is less than the Rayleigh resolution distance, and said detecting includes exposing the field with light of one wavelength, to obtain a diffraction image of PREs in the field, exposing the field with light of a second wavelength to obtain a second diffraction image of PREs in the field, and comparing the distance between peaks in the two diffraction patterns.
 16. The method of claim 1 , for use in interrogating a change in the environment of the field, wherein said discriminating includes comparing the values of the detected spectral characteristic of a PRE in the field before and after said change.
 17. The method of claim 16 , wherein the field is interrogated for changes in the dielectric constant of environment.
 18. The method of claim 1 , for use in detecting motion of PREs in the field, wherein said detecting includes detecting the centers of the diffraction patterns of the PREs in the image plane, as a function of time.
 19. Apparatus for use in the method of claim 1 , for interrogating a field having a plurality of PREs distributed therein, comprising an optical light source for illuminating the field, an optical detector for detecting a spectral emission characteristics of individual PREs and other light scattering entities in the field, when the field is illuminated by the light source, an image processor operatively connected to the detector for constructing, from signals received from the detector, a computer image of the positions and values of the spectral emission characteristic of individual PREs and such other light-scattering entities present in the field, discriminator means for discriminating PREs with a selected spectral signature from other light-scattering entities in the computer image, and output means for displaying information about the field based on the information about the selected PREs.
 20. The apparatus of claim 19 , wherein said light source includes a bright field/dark field lens for directing light onto the field.
 21. The apparatus of claim 19 , wherein said light source includes means for illuminating the field at each of a plurality of different wavelengths.
 22. The apparatus of claim 19 , wherein said detector is a two-dimensional photodetector array capable of detecting a spectral emission characteristic simultaneously from a plurality of illuminated PREs in an illuminated field.
 23. The apparatus of claim 19 , wherein said detector includes means for spectrally separating light emitted from the PREs, and said image processor operates to form a computer image of the positions and values of the emission spectral characteristic of individual PREs and such other light-scattering entities present in the field at each of a plurality of different emission wavelengths.
 24. The apparatus of claim 23 , wherein the optical detector includes a two-dimensional array of optical fibers whose output is aligned so as to constitute a line source that is sent into a grating or prism for responding to that line source, and a two-dimensional detector array for responding to the spread of spectral light of each fiber in said line source of detected light.
 25. The apparatus of claim 19 or 23 , which further includes means for moving said target in an x-y plane, relative to said light source, to successively illuminate individual light-scattering entities in the field.
 26. The apparatus of claim 19 , wherein said image processor operates to construct an image of PRE positions and, for each light-scattering entity in the field, values of a spectral characteristic selected from the group consisting of peak position, peak intensity, or peak width at half intensity of the spectral emission curve, peak halfwidth in the image plane, and polarization or angle of incidence response.
 27. The apparatus of claim 19 , wherein said image processor operates to construct an image of PRE positions and, for each light scattering entity in the field, a value of a spectral characteristic selected from the group consisting of fluorescence emission spectrum and Raman spectrum.
 28. The apparatus of claim 19 , wherein said discriminator means includes means for discriminating PREs with a selected spectral signature from all other light-scattering entities in the field, based on detected values, for each light-scattering entity in the field, of peak position, peak intensity, or peak width at half intensity of the spectral emission curve, peak halfwidth in the image plane, and polarization or angle of incidence response.
 29. The apparatus of claim 29 , wherein said discriminating is effective to discriminate for a selected type of PREs, those selected PREs which are interacting with one another and those which are not, or one selected type of PRE from another selected type of PRE in the field.
 30. A composition of plasmon resonant particles (PRPs) having one or more populations of PRPs, and characterized by: (a) the PRPs have a width at halfheight of less than 100 nm; (b) the PRPs in a single population are all within 40 nm of a defined wavelength; (c) at least 80% of the PRPs in the composition satisfying criterion (a) are in one or more of the populations and have a spectral emission wavelength in a single range selected from the group consisting of: (i) >700 nm; (ii) 400-700 nm; and (iii) <400 nm; and (d) each population has at most a 30% overlap in number of PRPs with any other population in the composition.
 31. The composition of claim 30 , wherein at least 80% of the PRPs in the composition are in one or more of the populations and have a spectral emission wavelength in the 400-700 nm wavelength range.
 32. The composition of claim 30 or 31 , wherein the particles have a composition selected from the group consisting of (i) a solid silver particle, (ii) a silver particle with a gold core, and (iii) a particle with a dielectric core and an outer silver shell of at least about 5 nm.
 33. The composition of claim 30 , wherein the particles have localized at their surfaces, one from the following group: (i) surface-attached ligands adapted to bind to ligand-binding sites on a target, where the ligand/ligand-binding sites are conjugate binding pairs, (ii) fluorescent molecules, (iii) Raman-active molecular entities, and (iv) a blocking reagent to prevent non-specific binding, and (v) a coating with functional groups for covalent coupling to the PRPs.
 34. The composition of claim 33 , wherein the surface localized ligand is one of a conjugate pair selected from the group of pairs consisting of antigen/antibody, hormone/receptor, drug/receptor, effector/receptor, enzyme/substrate, lipid/lipid binding agent and complementary nucleic acids strands.
 35. The composition of claims 33, which includes first and second populations of PRPs having first and second different surface localized molecules or entities.
 36. The composition of claim 35 , for use in identifying a target having first and second ligand-binding sites, wherein the first and second surface bound molecules are first and second ligands effective to bind to said first and second ligand-binding sites, respectively.
 37. The composition of claim 36 , wherein the first and second surface-localized molecules are oligonucleotides having sequences that are complementary to first and second proximate sequence regions of a target polynucleotide.
 38. The composition of claim 35 , wherein the first and second surface-localized entities are Raman-active molecular entities with different Raman spectral characteristics.
 39. The composition of claim 30 , having first and second populations of PRPs, each with a different shape, at least one of which is spherical or tetrahedral.
 40. A diagnostic method for use in detecting the presence of, or information about, a target having a molecular feature of interest, comprising contacting the target with one or more PREs having surface localized molecules, to produce an interaction between the molecular feature and the localized molecules, illuminating the target with an optical light source, and determining the presence of or information about the target by detecting a plasmon resonance spectral emission characteristic of one or more PREs after such interaction with the target.
 41. The method of claim 40 , wherein said target contains a ligand-binding site, the surface-localized molecule is one of a ligand/ligand-binding site conjugate pair selected from the group of pairs consisting of antigen/antibody, hormone/receptor, drug/receptor, effector/receptor, enzyme/substrate, lipid/lipid binding agent and complementary nucleic acids strands, said contacting produces a PRE/target complex, and said detecting includes detecting a plasmon resonance spectral emission characteristic of the complex.
 42. The method of claim 41 , wherein said contacting further includes the step of washing the field to remove PREs not bound to the target through a ligand/ligand-binding interaction.
 43. The method of claim 41 , wherein the target has at least two proximately spaced ligand-binding sites, and said complex includes at least two proximately spaced PREs that have a spectral emission signature different from that of PREs in the absence of binding to the target.
 44. The method of claim 43 , for determining the presence of a target having first and second proximately spaced ligand-binding sites, wherein said contacting includes reacting the target with first and second populations of PREs having surface-localized first and second ligands, respectively, for binding to the first and second ligand binding sites, respectively.
 45. The method of claim 44 , wherein the target is a polynucleotide having first and second adjacent base sequence regions, the ligand molecules on the first and second PREs are complementary to said first and second regions, and said contacting is carried out under conditions which allow surface-attached ligand molecules to hybridize with complementary-sequence regions of the target.
 46. The method of claim 41 , wherein the PRE(s) contain surface-localized fluorescent reporter molecules, and the interaction of a PRE with the target or with another PRE at the target is effective to detectably alter a plasmon-resonance induced spectral emission characteristic of the fluorescent molecules on the PRE.
 47. The method of claim 41 , wherein the PRE(s) contain surface-localized Raman reporter molecular entities, and the interaction of a PRE with the target or with another PRE at the target is effective to detectably alter a plasmon-resonance induced spectral emission characteristic of the Raman entities on the PRE.
 48. The method of claim 41 , wherein the target has multiple ligand-binding sites, the PREs bind to two or more of these sites and said detecting includes constructing a spatial map of the sites of PRE attachment to the target, which is indicative of the relative spacings of the ligand-binding sites in the target.
 49. The method of claim 48 , for use in mapping regions of known sequence in a target polynucleotide which is in a substantially extended condition, wherein the target is contacted with a plurality of PREs, each having different surface-attached oligonucleotides effective to hybridize to one of the know-sequence regions of the target, said contacting is carried out under conditions which allow the PRE's surface-attached oligonucleotides to hybridize with the target's selected base sequences, and said detecting includes (i) washing the field to remove unbound PREs, and (ii) mapping the relative positions of the bound PREs according to their spectral emission characteristics.
 50. The method of claim 40 , for resolving the distance between two closely spaced target sites, wherein said PREs have substantially the same peak wavelength, wherein said detecting includes detecting a composite spectral emission characteristic of the two PREs including shifts and broadening of single-particle spectral peaks and appearance of new peaks.
 51. The method of claim 40 , for resolving the distance between two closely spaced target sites, wherein said PREs have different peak wavelengths, wherein said detecting includes separately detecting the center of the diffraction peak of each particles at different illuminating light wavelengths.
 52. The method of claim 41 , wherein said target includes an array of different-sequence oligo- or polynucleotides, the array is contacted with one or more PREs having one or more surface-attached polynucleotides, said contacting is carried out under conditions which allow the PRE's surface-attached polynucleotides to hybridize with the target array oligo- or polynucleotides, and said detecting includes (i) washing the target to remove unbound PREs, and (ii) detecting a spectral emission characteristic of PREs at each region of the array.
 53. The method of claim 41 , wherein said target is a polynucleotide present as a separated band in an electrophoresis gel, said contacting is carried out by exposing the surface of the gel to PREs under hybridization conditions.
 55. The method of claim 41 , wherein the molecular feature of interest is a molecule which functions catalytically to break a bond between two atoms in a molecular chain, said PRE includes a pair of PREs linked by said chain, said contacting is carried out under conditions effective to cleave the molecular chain, and said detecting includes detecting the disappearance of linked PREs or the appearance of unlinked PREs.
 56. The method of claim 41 , for detecting the presence of a target polynucleotide sequence having first and second contiguous nucleotide sequences, said contacting includes adding to the target, under hybridization conditions, first and second PREs having surface-localized first and second oligonucleotide probes complementary to the first and second target sequences, respectively, and treating the resulting hybridization product with a ligase enzyme, and said detecting includes detecting the presence of linked PREs. 